ALBERT

Description: Abstract 5113 Photodynamic therapy (PDT) is a cancer therapeutic treatment that uses a compound called the “photosensitizer” and a particular type of visible light. When photosensitizers are exposed to a specific wavelength of light (600-800 nm), cytotoxic oxygen species are generated that kill cells (Dougherty, TJ et al., JNCI 90:889, 1998). Several clinical trials are currently underway to evaluate the use of PDT for a variety of cancers. A phase II study has been completed with photodynamic therapy in the treatment of patients with lymphoma or chronic lymphocytic leukemia. (NCT00054171). Recently, we have focused our attention about the properties of the photosensitizer Pheophorbide a (Pba), a chlorine, and its effects on different types of solid tumor cells (Rapozzi, V et al., Cancer Biol Ther 14:1318, 2009). The objective of the present study is to investigate the biochemical and molecular mechanisms by which PDT signals the B-NHL Raji lymphoma cell line (as model) and rendering the cells susceptible to both the cytotoxic mechanism of the tumor microenvironment in vivo or to the response to cytotoxic agents in vitro. We hypothesized that treatment of Raji cells with Pba/PDT in our in vitro system may result in the inhibition of resistance factors that regulate tumor cell responses to both chemotherapeutic and immunotherapeutic drugs. Our recent findings demonstrated that the constitutively overexpressed transcription factor Yin Yang 1 (YY1) regulates, in part, tumor cell resistance in lymphoma (Vega, MI et al., J Immun 175:2174, 2005). Accordingly, we examined whether treatment of Raji lymphoma cells with Pba/PDT will also result in the downregulation of YY1 expression and reverse resistance. The Raji cells were seeded at a cell density of 2×105/ml in Petri dishes. When the cells reached a 70% confluency, they were treated with different concentration (80-160-240 nM) of Pba for three hours in the dark and were then irradiated by an LED light source (640 nm at 12,7 mW for 9 min; 6.7 J/cm2). Following the light treatment, the cells were harvested at different times of incubation (18-36h) to assess apoptosis by the activation of caspase 3 using flow cytometry. In addition, different aliquots of cells were used to prepare slides for immunohistochemistry analyses. The results demonstrate that, indeed, treatment with Pba/PDT resulted in the inhibition of YY1 protein expression in Raji cells. By immunohistochemistry, PDT inhibited the basal nuclear and cytoplasmic expression of YY1 and resulted in weak cytoplasmic YY1 expression. The mechanism of YY1 inhibition might have been the result of PDT-mediated inhibition of NF-κB activity (Karmakar, S. et al., Neurosci lett 415: 242, 2007) since YY1 is transcriptionally regulated by NF-κB (Wang, H et al., Mol Cell Biol 67:4374, 2007). In addition, our preliminary findings demonstrate that treatment of drug-resistant tumor cells with PDT sensitizes the cells to drug-induced apoptosis. Overall, the data suggest that YY1 may be considered as a novel therapeutic target in PDT. Based on the findings here, we are currently examining the role of PDT in the dysregulation of the NF-κB/YY1/Snail/RKIP loop (Wu, K and Bonavida, B. Crit Rev Immun 29:241, 2009) that regulates cell survival and proliferation and resistance in lymphoma. (We acknowledge Doctors Oscar Stafsudd and Romaine Saxton for their assistance.) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3152 We have reported that treatment of B-NHL cell lines with rituximab resulted in the inhibition of the constitutively activated PI3K-AKT pathway (Suzuki et al., Oncogene 26:6184, 2007). Examination of the mechanism by which rituximab inhibits the PI3K/Akt pathway revealed that it induces the expression of the PI3K/Akt inhibitor PTEN (phosphatase and tensin homolog detected on chromosome 10). Time kinetic analysis indicated that the induction of PTEN occurs as early as 6 h post-rituximab treatment. The objective of this study is to delineate the molecular mechanism by which PTEN is induced by rituximab. We hypothesized that rituximab-induced inhibition of the constitutively activated NF-κB pathway, directly and indirectly through inhibition of the PI3K/Akt pathway, may result in the inhibition downstream of the PTEN transcription factors and repressors, Snail and Yin Yang 1 (YY1). Snail has been reported to repress the transcription of PTEN (Escriva, M et al., Mol Cell Biol 28:1528, 2008). Also, YY1 has been reported to positively regulate Snail transcription and expression (Palmer, MB et al., Mol Cancer Res 7:221, 2009). In addition, the induction of PTEN by rituximab also results, in a feed-back loop, in the suppression of YY1 and Snail and potentiates the induction of PTEN (Petriella et al, Cancer Biology Therapy, 8, 1389, 2009). This hypothesis was tested using the B-NHL Ramos cells, as model, for these studies. Treatment of Ramos with rituximab (20ug/ml for 16 hours) resulted in the inhibition of NF-κB, Snail, and YY1 and induction of PTEN expression as assessed by western. The direct role of Snail and YY1 in the suppression of PTEN expression was demonstrated in cells transfected with Snail or YY1 siRNA. The treated cells demonstrated significant induction of PTEN and, concomitantly, inhibition of the PI3K/Akt pathway. We have reported that rituximab sensitizes B-NHL cells to apoptosis by various chemotherapeutic drugs and demonstrated that inhibition of the PI3K/Akt pathway by various inhibitors mimics rituximab in the sensitization of the tumor cells to apoptosis by chemotherapeutic drugs (Suzuki et al., Oncogene 26:6184, 2007). The role of PTEN induction by rituximab in the sensitization of resistanr B-NHL cells to drug-apoptosis was demonstrated in cells pre-treated with rituximab (to induce PTEN) and then transfected with PTEN siRNA. The transfected cells were resistant to drug-induced apoptosis compared to the control siRNA treated cells. Altogether, the above findings demonstrate that rituximab-induced inhibition of the PI3K/Akt pathway is due, in part, to the induction of PTEN through rituximab-induced inhibition of the PTEN repressors Snail and YY1, downstream of NF-κB. Thus, the induction of PTEN by rituximab plays a major role in the reversal of tumor cell resistance to chemotherapeutic drugs. Further, the findings reveal that the dysregulated PI3K/Akt/NF-κB/Snail/YY1/PTEN loop in B-NHL cells can be interfered by rituximab. This interference leads to the inhibition of cell survival and reversal of resistance through sensitization to drugs. We propose that the gene products in this loop are potential novel therapeutic targets in the treatment of lymphoma. Disclosures: No relevant conflicts of interest to declare.