Peter Solár

Down-regulation of Bcl-2 and Akt induced by combination of photoactivated hypericin and genistein in human breast cancer cells.

Presented experiment considers combination of genistein and photodynamic therapy with hypericin with a view to achieve higher therapeutic outcome in human breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, both identified in our conditions as photodynamic therapy resistant. Since genistein is known to suppress Bcl-2 expression, we predicted that photodynamic therapy with hypericin might benefit from mutual therapeutic combination. In line with our expectations, combined treatment led to down-regulation of Bcl-2 and up-regulation of Bax in both cell lines as well as to suppression of Akt and Erk1/2 phosphorylation induced by photoactivated hypericin in MCF-7 cells. Although Akt and Erk1/2 phosphorylation was not stimulated by photodynamic therapy with hypericin in MDA-MB-231 cells, it was effectively suppressed in combination. Variations in cell death signaling favoring apoptosis were indeed accompanied by cell cycle arrest in G(2)/M-phase, activation of caspase-7, PARP cleavage and increased occurrence of cells with apoptotic morphology of nucleus. All these events corresponded with suppression of proliferation and significantly lowered clonogenic ability of treated cells. In conclusion, our results indicate that pre-treatment with tyrosine kinase inhibitor genistein may significantly improve the effectiveness of photodynamic therapy with hypericin in MCF-7 and MDA-MB-231 breast cancer cells.

Erythropoietin and Cancer: The Unintended Consequences of Anemia Correction

Until 1990, erythropoietin (EPO) was considered to have a single biological purpose and action, the stimulation of red blood cell growth and differentiation. Slowly, scientific and medical opinion evolved, beginning with the discovery of an effect on endothelial cell growth in vitro and the identification of EPO receptors (EPORs) on neuronal cells. We now know that EPO is a pleiotropic growth factor that exhibits an anti-apoptotic action on numerous cells and tissues, including malignant ones. In this article, we present a short discussion of EPO, receptors involved in EPO signal transduction, and their action on non-hematopoietic cells. This is followed by a more detailed presentation of both pre-clinical and clinical data that demonstrate EPO’s action on cancer cells, as well as tumor angiogenesis and lymphangiogenesis. Clinical trials with reported adverse effects of chronic erythropoiesis-stimulating agents (ESAs) treatment as well as clinical studies exploring the prognostic significance of EPO and EPOR expression in cancer patients are reviewed. Finally, we address the use of EPO and other ESAs in cancer patients.

Differentially expressed genes associated with cisplatin resistance in human ovarian adenocarcinoma cell line A2780

Ovarian cancer cells are usually initially sensitive to platinum-based chemotherapy, such as cisplatin (CDDP), but typically become resistant over time. Such drug resistance is a serious impediment to successful disease treatment, and the molecular mechanisms responsible for resistance are not fully understood. In search of novel mechanisms that may lead to the development of CDDP chemoresistance, we used subtractive hybridization to identify differentially expressed genes in CDDP resistant CP70 and C200 cells vs. CDDP sensitive A2780 human ovarian adenocarcinoma cells. We analyzed 256 randomly selected clones. Subtraction efficiency was determined by dot blot and DNA sequencing. Confirmation of differentially expressed cDNAs was done by virtual northern blot analysis, and 17 genes that were differentially expressed in CDDP resistant cell lines vs. CDDP sensitive A2780 cells were identified. The expression of 10 of these genes was low or undetectable in sensitive A2780 cells in comparison to resistant cells and an additional seven genes were more highly expressed in resistant CP70 and C200 vs. A2780 cells. Our identified genes are involved in numerous and diverse cellular processes, such as inhibition of apoptosis (ARHGDIB), stress response (HSPCA, TRA1), chromatin condensation (CNAP1, RanBP2), invasiveness of cells (MMP10), alteration of Ca(2+) homeostasis (ASPH, ATP2B1) and others. Further characterization of these genes and gene products should yield important insights into the biology of CDDP resistance in ovarian carcinoma.

Hsp90 inhibitor as a sensitizer of cancer cells to different therapies (Review)

Hsp90 is a molecular chaperone that maintains the structural and functional integrity of various client proteins involved in signaling and many other functions of cancer cells. The natural inhibitors, ansamycins influence the Hsp90 chaperone function by preventing its binding to client proteins and resulting in their proteasomal degradation. N- and C-terminal inhibitors of Hsp90 and their analogues are widely tested as potential anticancer agents in vitro, in vivo as well as in clinical trials. It seems that Hsp90 competitive inhibitors target different tumor types at nanomolar concentrations and might have therapeutic benefit. On the contrary, some Hsp90 inhibitors increased toxicity and resistance of cancer cells induced by heat shock response, and through the interaction of survival signals, that occured as side effects of treatments, could be very effectively limited via combination of therapies. The aim of our review was to collect the data from experimental and clinical trials where Hsp90 inhibitor was combined with other therapies in order to prevent resistance as well as to potentiate the cytotoxic and/or antiproliferative effects.

Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells

Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin. [Mol Cancer Ther 2008;7(8):2263–71]

Modulation of Hypericin Photodynamic Therapy by Pretreatment with 12 Various Inhibitors of Arachidonic Acid Metabolism in Colon Adenocarcinoma HT-29 Cells

One proposal to increase the efficiency of photodynamic therapy (PDT) is to accompany photosensitization with other treatment modalities, including modulation of arachidonic acid (AA) metabolism. The aim of this study was to evaluate the effectiveness of a combined modality approach employing 48 and 24 h pretreatment with various inhibitors of lipoxygenase (LOX; nordihydroguaiaretic acid, esculetin, AA‐861, MK‐886 and baicalein), cyclooxygenase (COX; diclofenac, flurbiprofen, ibuprofen, indomethacin, SC‐560 and rofecoxib) and cytochrome P450‐monooxygenase (proadifen) pathways, followed by hypericin‐mediated PDT. Cytokinetic parameters like MTT assay, adherent and floating cell numbers, viability and cell cycle distribution analysis were examined 24 h after hypericin activation. Pretreatment of human colon cancer cells HT‐29 prior to PDT with 5‐LOX inhibitor MK‐886 as well as 5, 12‐LOX and 12‐LOX inhibitors (esculetin and baicalein, respectively) resulted in significant and dose‐dependent effects on all parameters tested. Pretreatment with diclofenac, flurbiprofen, ibuprofen and indomethacin, the nonspecific COX inhibitors, promoted hypericin‐mediated PDT, but these effects were probably COX‐independent. In contrast, application of SC‐560 and rofecoxib, specific inhibitors of COX‐1 and COX‐2, respectively, attenuated PDT. Inhibition of P450 monooxygenase with proadifen implied also the significance of this metabolic pathway in cell survival and cell resistance to hypericin photocytotoxicity. In conclusion, our results testify that application of diverse inhibitors of AA metabolism may have different consequences on cellular response to hypericin‐mediated PDT and that some of them could be considered for potentiation of PDT.

Degradation of HER2 Receptor Through Hypericin‐mediated Photodynamic Therapy

Current treatment of breast cancer is often affected by resistance to therapeutics, for which the human epidermal growth factor receptor 2 (HER2) may be responsible. Here, we report for the first time the use of hypericin‐mediated photodynamic therapy (HY‐PDT) in combination with a selective HER2 inhibitor (AG 825) on SKBR‐3, a HER2 overexpressing human breast adenocarcinoma cell line. The results demonstrate that HY‐PDT is able to degrade HER2 with an impact on its signaling cascade. Combination with AG 825 resulted in increased apoptosis induction, total degradation of HER2 and inhibition of colony formation. Downregulation of HSP90, Mcl‐1, Bcl‐xL and upregulation of Bax was also observed. This knowledge provides the basis for the possible application of HY‐PDT in preclinical and clinical models of breast cancer treatment.

Photoactivated Hypericin Induces Downregulation of HER2 Gene Expression

Abstract Photodynamic therapy is an alternative method for cancer treatment in which a photosensitizer exposed to a light source of suitable wavelength is excited and can subsequently react through free radical mechanisms. Recently, oxygen free radical-mediated changes in gene expression have been established. The present study shows the effect of photoactivated hypericin on the expression of the human epidermal growth factor receptor 2 (HER2) oncogene at both the mRNA and the protein level in SKBR-3 and MCF-7 breast adenocarcinoma cell lines. The photodynamic therapy-induced decrease in mRNA expression was reversed by the singlet oxygen scavenger trolox, which supports a role for singlet oxygen. In addition, prevention of the generation of reactive oxygen species by pretreatment with trolox effectively blocked the antiproliferation activity of photoactivated hypericin. These results may have important implications at least for recurrent breast cancer with HER2 expression alone or in combination with conventional therapies.

Association between polymorphisms of XRCC1, p53 and MDR1 genes, the expression of their protein products and prognostic significance in human breast cancer

Summary Background This study aimed to examine the relationship between XRCC1, p53 and MDR1 protein, along with polymorphisms of their genes and their prognostic values in breast cancer. The following clinical and pathological parameters were evaluated: histopathological type of tumor, grade, stage, Her2/neu expression, ER, PR positivity and involvement of regional lymph nodes. Material/Methods Expression of proteins was determined in 39 samples of breast cancer by immunohistochemistry. Nucleotide polymorphisms were analyzed by PCR-RFLP. For statistical analysis, chi-square test (Yates), Fisher’s exact test, and correlation test were used to analyze the data. Results The highest protein expression was immunohistochemically found in MDR1 protein, with 54% of samples testing positive. In addition, the evaluation of MDR1 expression revealed higher positive immunoreactivity in lobular (LIC) and other types of tumor in comparison to ductal (DIC) type. The expression of p53 and XRCC1 protein was equal, but lower compared to MDR1, both testing positive in 36% of all tissue samples. Comparison of XRCC1 protein and histopathological type of tumor revealed that DIC and LIC types were mostly XRCC1-negative, while other types, papillary and mucinous were more likely to be XRCC1-positive. Interestingly, when evaluating LIC samples separately, a negative correlation between the Her2/neu and expression of XRCC1 was detected. Apparently, all Her2/neu-positive samples were XRCC1-negative (6/86%). The correlation test indicated a negative correlation between Her2/neu-positive samples and XRCC1-negative specimens (r=1, p<0.05). Statistical analysis did not reveal a correlation of p53 expression with clinical and pathological parameters. Similarly, no statistically significant difference was found between the tested polymorphisms and protein expression. Conclusions We did not find statistically significant correlation between tested polymorphisms and their protein expression.

Antitumor effects of atorvastatin in the chemoprevention of rat mammary carcinogenesis

Epidemiological studies indicate that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, play a role in inhibition of several human neoplasia including breast cancer. In this study, chemopreventive effects of atorvastatin in N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. Atorvastatin was administered in the diet at two concentrations: 10 mg/kg (ATOR 10) and 100 mg/kg (ATOR 100). Atorvastatin treatment began 8 days prior to carcinogen administration and subsequently continued for 15 weeks till the end of the experiment. Atorvastatin at a higher dose suppressed tumor frequency by 80.5% (P = 0.0008) and tumor incidence by 49.5% (P = 0.015), and extended latency period by 14 days (P = 0.076) when compared to the control group. Atorvastatin at a lower dose did not significantly alter tumor parameters in comparison with the control group. In the specimens of mammary tumors, atorvastatin (in the ATOR 100 group) significantly decreased mRNA expression of Bcl-2 gene but non-significantly increased Bax mRNA expression compared to control group. Atorvastatin administration did not alter serum concentration of triacylglycerols, total cholesterol, and LDL cholesterol in comparison with controls. This study is the first report on tumor suppressive effect of atorvastatin in rat mammary carcinogenesis.