The Immune Response of Cancer Cells in Breast and Gynecologic Neoplasms.

Cancer diseases constitute a major health problem which leads to the death of millions of people annually. They are unique among other diseases because cancer cells can perfectly adapt to the environment that they create themselves. This environment is usually highly hostile and for normal cells it would be hugely difficult to survive, however neoplastic cells not only can survive but also manage to proliferate. One of the reasons is that they can alter immunological pathways which allow them to be flexible and change their phenotype to the one needed in specific conditions. The aim of this paper is to describe some of these immunological pathways that play significant roles in gynecologic neoplasms as well as review recent research in this field. It is of high importance to possess extensive knowledge about these processes, as greater understanding leads to creating more specialized therapies which may prove highly effective in the future.

Natural Compounds in Non-Melanoma Skin Cancer: Prevention and Treatment.

The elevated occurrence of non-melanoma skin cancer (NMSC) and the adverse effects associated with available treatments adversely impact the quality of life in multiple dimensions. In connection with this, there is a necessity for alternative approaches characterized by increased tolerance and lower side effects. Natural compounds could be employed due to their safety profile and effectiveness for inflammatory and neoplastic skin diseases. These anti-cancer drugs are often derived from natural sources such as marine, zoonotic, and botanical origins. Natural compounds should exhibit anti-carcinogenic actions through various pathways, influencing apoptosis potentiation, cell proliferation inhibition, and metastasis suppression. This review provides an overview of natural compounds used in cancer chemotherapies, chemoprevention, and promotion of skin regeneration, including polyphenolic compounds, flavonoids, vitamins, alkaloids, terpenoids, isothiocyanates, cannabinoids, carotenoids, and ceramides.

Effect of Interaction between Chromium(VI) with 17ß-Estradiol and Its Metabolites on Breast Cancer Cell Lines MCF-7/WT and MDA-MB-175-VII: Preliminary Study.

The number of factors initiating and stimulating the progression of breast cancer are constantly increasing. Estrogens are a risk factor for breast adenocarcinoma, the toxicity of which increases as a result of metabolism and interaction with other factors. Due to the presence of environmental exposure to estrogens and metalloestrogens, we investigated how interactions between estrogens and toxic chromium(VI)[Cr(VI)] affect breast cancer lines and investigated whether estrogens play a protective role. The aim of the study was to investigate the effect of 17ß-estradiol and its metabolites: 2-methoxyestradiol (2-MeOE2), 4-hydroxyestradiol (4-OHE2), and 16α-hydroxyestrone (16α-OHE1) in exposure to Cr(VI) on cell viability and DNA cell damage. Two estrogen-dependent breast cancer cell lines, MCF 7/WT and MDA-MB-175-VII, were examined. In addition, the expression of Cu-Zn superoxide dismutase (SOD1) was determined immunocytochemically to elucidate the mechanism of oxidative stress. The effects of single substances and their mixtures were tested in the model of simultaneous and 7-day estrogen pre-incubation. As a result, the viability of MCF-7 and MDA-MB-175-VII cells is lowered most by Cr(VI) and least by 17ß-E2. In the combined action of estrogens and metalloestrogens, we observed a protective effect mainly of 17ß-E2 against Cr(VI)-induced cytotoxicity. The highest expression of SOD1 was found in MCF-7/WT cells exposed to 17ß-E2. Moreover, high apoptosis was caused by both Cr(VI) itself and its interaction with 4-OHE2 and 2-MeOE2. The direction and dynamics of changes in viability are consistent for both lines.

Betulin and Its Derivatives Reduce Inflammation and COX-2 Activity in Macrophages.

Betulin is a heavily studied natural compound for its use as an anticancer or pro-regenerative agent. The structural similarity between betulin to steroids gives rise to the idea that the substance may as well act as an anti-inflammatory drug. This study is the first to describe the anti-inflammatory properties of betulinic acid, betulin, and its derivatives with amino acids 1,4-diaminebutane (Dab), 1,3-diaminepropane (Dap), Ornithine (Orn), and lysine (Lys) on murine macrophages from lymphoma site. The compounds were compared to dexamethasone. To establish the response of the macrophages to the natural compounds, we tested the viability as well as sensitivity to the inflammatory signaling (IFNγR). IL-6 secretory properties and HSP-70 content in the cells were examined. Furthermore, we characterized the effects of compounds on the inhibition of cyclooxygenase-2 (COX-2) activity both in the enzymatic assays and molecular docking studies. Then, the changes in COX-2 expression after betulin treatment were assessed. Betulin and betulinic acid are the low-cytotoxicity compounds with the highest potential to decrease inflammation via reduced IL-6 secretion. To some extent, they induce the reorganization of IFNγR with nearly no effect on COX-2 activity. Conversely, Bet-Orn and Bet-Lys are highly cytotoxic and induce the aggregation of IFNγR. Besides, Bet-Lys reduces the activity of COX-2 to a higher degree than dexamethasone. Bet-Orn is the only one to increase the HSP-70 content in the macrophages. In case of IL-6 reduction, all compounds were more potent than dexamethasone.

Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance.

Nanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which oxidative stress is a principal component. In the present study, nsPEF-induced oxidative stress was investigated in two gastric adenocarcinoma cell lines (EPG85-257P and EPG85-257RDB) which differ by their sensitivity to daunorubicin. Cells were exposed to 200 pulses of 10 ns duration, with the amplitude and pulse repetition frequency at 1 kHz, with electric field intensity varying from 12.5 to 50 kV/cm. The electroporation buffer contained either 1 mM or 2 mM calcium chloride. CellMask DeepRed visualized cell plasma permeabilization, Fluo-4 was used to visualize internal calcium ions content, and F-actin was labeled with AlexaFluor®488 for the cytoskeleton. The cellular viability was determined by MTT assay. An alkaline and neutral comet assay was employed to detect apoptotic and necrotic cell death. The luminescent method estimated the modifications in GSSG/GSH redox potential and the imbalance of proteasomal activity (chymotrypsin-, trypsin- and caspase-like). The reactive oxygen species (ROS) level was measured by flow cytometry using dihydroethidium (DHE) dye. Morphological visualization indicated cell shrinkage, affected cell membranes (characteristic bubbles and changed cell shape), and the reorganization of actin fibers with sites of its dense concentration; the effect was more intense with the increasing electric field strength. The most significant decrease in cell viability and GSSG/GSH redox potential was noted at the highest amplitude of 50 kV/cm, and calcium ions amplified this effect. nsPEF, particularly with calcium ions, inhibited proteasomal activities, resulting in increased protein degradation. nsPEF increased the percentage of apoptotic cells and ROS levels. The EPG85-257 RDB cell line, which is resistant to standard chemotherapy, was more sensitive to applied nsPEF protocols. The applied nsPEF method disrupted the metabolism of cancer cells and induced apoptotic cell death. The nsPEF ability to cause apoptosis, oxidative stress, and protein degradation make the nsPEF methodology a suitable alternative to current anticancer pharmacological methods.

Synthesis, Anticancer Activity and Molecular Docking Studies of Novel N-Mannich Bases of 1,3,4-Oxadiazole Based on 4,6-Dimethylpyridine Scaffold.

Cancer is one of the greatest challenges in modern medicine today. Difficult and long-term treatment, the many side effects of the drugs used and the growing resistance to treatment of neoplastic cells necessitate new approaches to therapy. A very promising targeted therapy is based on direct impact only on cancer cells. As a continuation of our research on new biologically active molecules, we report herein the design, synthesis and anticancer evaluation of a new series of N-Mannich-base-type hybrid compounds containing morfoline or different substituted piperazines moieties, a 1,3,4-oxadiazole ring and a 4,6-dimethylpyridine core. All compounds were tested for their potential cytotoxicity against five human cancer cell lines, A375, C32, SNB-19, MCF-7/WT and MCF-7/DX. Two of the active N-Mannich bases (compounds 5 and 6) were further evaluated for growth inhibition effects in melanoma (A375 and C32), and normal (HaCaT) cell lines using clonogenic assay and a population doubling time test. The apoptosis was determined with the neutral version of comet assay. The confocal microscopy method enabled the visualization of F-actin reorganization. The obtained results demonstrated that compounds 5 and 6 have cytotoxic and proapoptotic effects on melanoma cells and are capable of inducing F-actin depolarization in a dose-dependent manner. Moreover, computational chemistry approaches, molecular docking and electrostatic potential were employed to study non-covalent interactions of the investigated compounds with four receptors. It was found that all the examined molecules exhibit a similar binding affinity with respect to the chosen reference drugs.

Mechanisms of curcumin-based photodynamic therapy and its effects in combination with electroporation: An in vitro and molecular dynamics study.

Photodynamic therapy (PDT) and electrochemotherapy (ECT) are two methods designed to enhance the anticancer potential of various drugs. Various clinical trials proved the efficacy of both ECT and PDT in melanoma treatment. Curcumin is a natural polyphenolic compound with high anticancer potential against melanoma due to its light absorption properties and toxicity towards cancer cells; however, high reactivity and amphipathic structure of curcumin are limiting its utility. This study aimed to propose the most effective protocol for antimelanoma combination of both therapies (PDT and ECT) in the context of curcumin. The in vitro studies were carried on melanotic melanoma (A375), amelanotic melanoma (C32) and fibroblast (HGF) cell lines. In molecular dynamics studies curcumin presented the single-layer localization in the water-membrane interphase. Further, the mass spectrometry studies exposed that during the PDT treatment curcumin is degraded to vanillin, feruloylmethane, and ferulic acid. Instant ECT with curcumin followed by PDT is the most efficient approach due to its selective genotoxicity towards malignant cells. The metabolic activity of fibroblasts decreased, however, at the same time the fragmentation of DNA did not occur. Additionally, instant PDT with curcumin followed by ECT after 3 h of incubation was a therapy selective towards melanotic melanoma.

Effects of curcumin based PDT on the viability and the organization of actin in melanotic (A375) and amelanotic melanoma (C32) - in vitro studies.

Curcumin is a turmeric, antioxidative compound, well-known of its anti-cancer properties. Nowadays more and more effort is made in the field of enhancing the efficiency of the anticancer therapies. Combining the photoactive properties of curcumin with the superficial localization of melanoma and photodynamic therapy (PDT) seems to be a promising treatment method. The research focused on the evaluation of the curcumin effectiveness as an anticancer therapeutic agent in the in vitro treatment of melanotic (A375) and amelanotic (C32) melanoma cell lines. Keratinocytes (HaCat) and fibroblasts (HGF) were used to assess the impact of the therapy on the skin tissue. The aim of the study was to investigate the cell death after exposure to light irradiation after preincubation with curcumin. Additionaly the authors analized the interactions between curcumin and the actin cytoskeleton. The cytotoxic effect initiated by curcumin and increased by irradiation confirm the usefulness of the flavonoid in the PDT approach. Depending on curcumin concentration and incubation time, melanoma cells survival rate ranged from: 93.68 % (C32 cell line, 10 µM, 24 h) and 83.47 % (A375 cell line, 10 µM, 24 h) to 8.98 % (C32 cell line, 50 µM, 48 h) and 12.42 % (A375 cell line, 50 µM, 48 h). Moreover, photodynamic therapy with curcumin increased the number of apoptotic and necrotic cells in comparison to incubation with curcumin without irradiation. The study demonstrated that PDT induced caspase-3 overexpression and DNA cleavage in the studied cell lines. The cells revealed decreased proliferation after the therapy due to the actin cytoskeleton rearrangement. Although effective, the therapy remains not selective towards melanoma cells.

3PO as a Selective Inhibitor of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 in A375 Human Melanoma Cells.

BACKGROUND/AIM: The occurrence of BRAFV600E mutation causes an up-regulation of the B-raf kinase activity leading to the stabilization of hypoxia-inducible factor 1-alpha (HIF-1α) - the promoter of the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) enzyme. The aim of the study was to examine the effect of the (2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), as an inhibitor of PFKFB3, on human melanoma cells (A375) with endogenous BRAFV600E mutation. MATERIALS AND METHODS: A375 cells were exposed to different concentrations of 3PO and the following tests were performed: docking, cytotoxicity assay, immunocytochemistry staining glucose uptake, clonogenic assay, holotomography imaging, and flow cytometry. RESULTS: Our studies revealed that 3PO presents a dose-dependent and time-independent cytotoxic effect and promotes apoptosis of A375 cells. Furthermore, the obtained data indicate that 3PO induces cell cycle arrest in G1/0 and glucose uptake reduction. CONCLUSION: Taking all together, our research demonstrated a here should be proapoptotic and antiproliferative effect of 3PO on A375 human melanoma cells.

Proapoptotic activity induced by photodynamic reaction with novel cyanine dyes in caspase-3-deficient human breast adenocarcinoma cell lines (MCF/WT and MCF/DX).

Photodynamic therapy (PDT) is currently one of the cancer treatment options. PDT requires the application of a photosensitizer (such as: porphyrins, chlorines, and phthalocyanines) that selectively targets malignant cells. It is a dilemma to find a proper photosensitizer. In our study, we have tested a new in-vitro group of cyanine dyes. These dyes are widely applied in biotechnology as fluorescent markers. Two malignant adenocarcinoma cell lines (MCF-7/WT and MCF-7/DOX) were investigated using photodynamic reaction (PDR) with four cyanine dyes (KF-570, HM-118, FBF-749, and ER-139). KF-570 and HM-118 were irradiated with red light (630 nm), whereas FBF-749 and ER-139 with green light (435 nm). To evaluate PDR efficiency, a clonogenic test was conducted. Apoptosis was investigated by TUNEL and NCA (neutral comet) assays. Proteins selected as indicators of the apoptotic pathway (AIF, sPLA2, Smac/Diablo) and intracellular response markers (SOD-1 and GST-pi) were detected using western blot. The highest number of apoptotic cells (ca. 100%) was observed after PDR with HM-118 and KF-570 in both conducted tests, in both cell lines. The results showed that HM-118 and KF-570 cyanine dyes demonstrated a major phototoxic effect causing apoptosis in doxorubicin-resistant and sensitive cell lines.

Electropermeabilization of metastatic chondrosarcoma cells from primary cell culture.

Primary cell cultures are challenging, but reliable model reflecting tumor response in vitro. The study was designed to examine if the increased electropermeabilization can overcame initial drug insensitivity in chondrosarcoma cells from lung metastasis. We established a primary cell culture and evaluated the cytotoxic impact of four drugs-cisplatin (CDDP), camptothecin, 2-methoxyestradiol, and leucovorin calcium (LeuCa). After determination of parameters allowing for electropermeabilization, we performed electrochemotherapy in vitro with the least toxic drugs-CDDP and LeuCa. Although combining CDDP and leucovorin together increased their toxicity and supported apoptosis, application of pulsed electric fields (PEFs) brought no advantage for their efficacy. The study emphasizes the need for introduction of primary cell cultures into studies on pulse electric fields as model frequently less sensitive to PEF-based treatments than continuous cell lines.

The Cytoprotective Role of Antioxidants in Mammalian Cells Under Rapidly Varying UV Conditions During Stratospheric Balloon Campaign.

The current age of dynamic development of the space industry brings the mankind closer to routine manned space flights and space tourism. This progress leads to a demand for intensive astrobiological research aimed at improving strategies of the pharmacological protection of the human cells against extreme conditions. Although routine research in space remains out of our reach, it is worth noticing that the unique severe environment of the Earth's stratosphere has been found to mimic subcosmic conditions, giving rise to the opportunity to use the stratospheric surface as a research model for the astrobiological studies. Our study included launching into the stratosphere a balloon containing mammalian normal and cancer cells treated with various compounds to examine whether these substances are capable of protecting the cells against stress caused by rapidly varying temperature, pressure, and radiation, especially UV. Owing to oxidative stress caused by irradiation and temperature shock, we used natural compounds which display antioxidant properties, namely, catechin isolated from green tea, honokiol derived from magnolia, curcumin from turmeric, and cinnamon extract. "After-flight" laboratory tests have shown the most active antioxidants as potential agents which can minimize harmful impact of extreme conditions on human cells.

A New Betulin Derivative Stimulates the Synthesis of Collagen in Human Fibroblasts Stronger than its Precursor.

BACKGROUND/AIM: The exploration of substances that stimulate collagen synthesis and retard the aging process of the skin is an active field of current research. The natural environment and plants used in traditional medicine have been a source of such substances. The aim of this study was to compare the stimulatory effect of betulin (BE), betulinic acid (BA) and the new derivative - betulin ester with diaminobutyl acid (BE-Dab-NH2) on collagen synthesis in human normal fibroblasts. MATERIALS AND METHODS: Primary fibroblast cultures were obtained from the gums of a healthy patient. The effect of the above-mentioned compounds was assessed by Sircol collagen assay, immunocytochemistry, and proliferation test. RESULTS: Fibroblasts cultured in the presence of BE-Dab-NH2 produced 6.85 times more collagen than control cells, 7.85 times more than those cultured in the presence of BA and 6.31 times more than those cultured in the presence of BE. An intense immunocytochemical reaction for collagen type I and III was found in fibroblasts cultured in the presence of BE-Dab-NH2 Conclusion: BE-Dab-NH2 stimulates significantly more collagen synthesis in normal human fibroblasts than its precursor.

Cisplatin and Vinorelbine -Mediated Electrochemotherapeutic Approach Against Multidrug Resistant Small Cell Lung Cancer (H69AR) In Vitro.

BACKGROUND/AIM: Small cell lung cancer (SCLC) originates from neuroendocrine branchial cells (15-20%). It is regarded as distinct from other lung cancers due to its biological and clinical features. In most cases of SCLC, surgery or radiotherapy alone is not an effective cure. The aim of our study was to examine the cytotoxic effects of chemotherapy supported by electroporation (EP) on a resistant SCLC model, in vitro. MATERIAL AND METHODS: The multidrug resistant small lung cell line H69AR was used to evaluate the cytotoxic effects of cisplatin (CPPD) and vinorelbine (Navirel®; NAV) at lower doses when used with EP. Cells were treated with different concentrations of CPPD and NAV, alone or in combination with the following EP parameters: 400-1200 V/cm, 8 pulses of 100 µs duration, at 1Hz. The cell viability was estimated by MTT assay after 24 and 48 h. Apoptotic cells were detected by neutral comet assay and immunofluorescence assay with PARP-6. RESULTS: CPPD and NAV alone showed a dose-dependent effect on cell viability. Cytostatic drugs combined with EP revealed increased anticancer activity. Lower doses of CPPD or NAV delivered by EP were as effective as higher doses of these drugs without EP. The electrochemotherapeutic protocols increased the number of apoptotic cells and increased immunoreactivity of PARP-6. Our results indicated higher sensitivity of H69AR cells to NAV supported by EP. CONCLUSION: In SCLC cells, an increased anticancer activity was potentiated by exposure of cells to high intensity electric pulses and low drug doses. It is suggested that this method could be effectively applied in the treatment of lung cancer.

Valspodar-modulated chemotherapy in human ovarian cancer cells SK-OV-3 and MDAH-2774.

Overcoming drug resistance in ovarian cancer is the overarching goal in gynecologic oncology. One way to increase drug cytotoxicity without increasing the drug dose is to simultaneously apply multidrug resistance modulator. Valspodar is the second generation P-glycoprotein 1 modulator capable of reversing multidrug resistance in different cancers. In this study, we evaluated the effect of valspodar and cisplatin co-treatment on cell viability, cell death and oxidative status in ovarian cancer cells. Two human ovarian cancer cell lines SK-OV-3 and MDAH-2774 were treated with cisplatin, valspodar, or cisplatin + valspodar for 24 or 48 hours. Untreated cells were used as control group. Cell viability was evaluated by MTT assay. Cell death was assessed by TUNEL and comet assay. Lipid peroxidation (malondialdehyde) and protein thiol groups were analyzed as oxidative stress markers. The expression of mitochondrial superoxide dismutase (MnSOD) was assessed by immunocytochemistry. Valspodar effectively reduced the resistance of SK-OV-3 cells to cisplatin, as demonstrated by increased oxidative stress, decreased cell viability and increased apoptosis in SK-OV-3 cells co-treated with valspodar and cisplatin compared to other groups. However, valspodar did not significantly affect the resistance of MDAH-2774 cells to cisplatin. Stronger staining for MnSOD in MDAH-2774 vs. SK-OV-3 cells after co-treatment with cisplatin and valspodar may determine the resistance of MDAH-2774 cell line to cisplatin.

Anticancer properties of ester derivatives of betulin in human metastatic melanoma cells (Me-45).

BACKGROUND: Betulinic acid and betulin are triterpenes that have anticancer properties in various types of cancer. Unfortunately, the bioavailability and the bio-distribution of betulinic acid and its metabolic precursor, betulin are very low because of poor solubility in aqueous buffers. METHODS: In this study, we examined the anticancer properties of the ester derivatives of betulin compared to their precursors in a malignant melanoma cell line. We assessed five amino acid esters of betulin. The compounds contained four basic amino acids-natural lysine (l-Lys-OH) and three of its derivatives (l-Dap-OH, l-Dab-OH, and l-Orn-OH)-and alanine (l-Ala-OH) as a negative control (amino acid without an amine group in the side chain). The derivatives were more soluble than their precursors (betulin and betulinic acid) in water. The betulin esters were tested in the malignant melanoma cell line Me-45. To evaluate the cytotoxicity, MTT test was performed after 24, 48 and 72 h of incubation with the test compounds at a concentration range of 0.75-100 µM. For analysis of the apoptotic activity, TUNEL assay was performed. Additionally, expression of caspase-3 and PARP-1 was investigated immunocytochemically. RESULTS: The highest biological activity was observed with the lysine ester. The results showed that the highest cytotoxicity and the highest number of positively stained nuclei in metastatic melanoma Me-45 cells were obtained after 72 h of incubation with betulin derivatives containing lysine and ornithine. CONCLUSIONS: The betulin ester derivatives showed enhanced antitumor activity compared to their non-modified precursors. Esters of betulin can be more potent anticancer agents than their precursor as a consequence of the rapid bioavailability and increased concentration in cancer cells.

Classical and atypical resistance of cancer cells as a target for resveratrol.

The phenomenon of cancer cell resistance to chemotherapeutics is the main cause of insensitivity to anticancer therapy. Thus, the current challenge remains searching for substances sensitising the activity of cytostatic drugs. In this respect, resveratrol is a very promising therapeutic agent. It has pleiotropic effect on cancer cells, which can play a key role in numerous resistance mechanisms, both classical and atypical. The purpose of the present study was to assess the effect of resveratrol on the inhibition of human pancreatic cancer cell proliferation and on the level of cytostatic resistance-associated proteins. The study was performed on human pancreatic cancer cell lines EPP85-181P (control), EPP85-181RDB (daunorubicin resistance) and EPP85-181PRNOV (mitoxantrone resistance). The effect of resveratrol on the viability and proliferation of the studied cell lines was evaluated by SRB assay, whereas cell cycle arrest and cytostatic accumulation by FACS. Western blot analysis was used to determine the level of P-glycoprotein, topoisomerase II α and ß and immunofluorescence technique to visualise the proteins in the cells. Resveratrol inhibited proliferation of all studied cell lines. Phase-specific cell cycle arrest depended on the type of cancer cells. Resveratrol decreased the level and activity of P-gp in EPP85-181RDB cells. In EPP85-181PRNOV cells, expression of both TopoII isoforms increased in a statistically significant manner. The results of in vitro studies support the possibility of potential use of resveratrol in breaking cancer cell resistance to chemotherapeutic drugs.

Plasminogen Activation System in Rectal Adenocarcinoma.

BACKGROUND: The purpose of the present study was to determine the extent of expression of proteins that are the elements of the plasminogen activation system, i.e. urokinase plasminogen activator (uPA), receptor of uPA (uPAR), plasminogen activating inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in the primary site of rectal adenocarcinoma, as well as in lymph node metastases, and their correlation with clinical and pathological data. MATERIALS AND METHODS: The study material consisted of archival paraffin blocks from 108 patients with confirmed oncological diagnosis, treated in 2002 and 2003 in the Lower Silesian Oncology Centre in Wroclaw. This material was used for immunohistochemical reactions with antibodies against uPA, uPAR, PAI-1 and tPA. The extent of expression was evaluated semi-quantitatively based on the immunoreactive score according to Remmele and Stegner. The obtained results were correlated with clinical and pathological data: stage of the disease (modified Dukes' classification of Astler and Coller), grade of histological malignancy, event-free survival and overall survival. RESULTS: It was found that cytoplasmic expression of uPA, uPAR, PAI-1 and tPA was higher in cells of tumor and metastases of perienteric lymph nodes in comparison to normal rectal tissues. Positive correlation was shown between expression of PAI-1 and tPA, uPA and uPAR, as well uPA and PAI-1 (r=0.70, r=0.77 and r=0.34, respectively; p<0.05 for all). Additionally, in patients with low and moderate expression of uPA and uPAR, the overall survival rate was higher in comparison to patients with high expression of the studied markers. CONCLUSION: The intensity of expression of uPA and uPAR might be a prognostic factor of survival time for patients with primary rectal adenocarcinomas.

The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment - In vitro studies.

BACKGROUND: Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications. OBJECTIVE: To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells. METHODS: In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids - natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, -CH2-CH2-CH2-CH2-NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (-CH2-CH2-CH2-NH2)>Dab (-CH2-CH2-NH2)>Dap (-CH2-NH2)>Ala (CH3)>betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72h of incubation with the test compounds at a concentration range of 0.75-100µM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically. RESULTS: The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50=7µM) and ornithine (IC50=10µM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain. CONCLUSIONS: The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.