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The aim of the present study was to investigate the effects of betulinic acid (BetA) on the expression and distribution pattern of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), an indirect indicator of nitric oxide (NO) synthase in the thymus and spleen of mice. Mice were randomly assigned to four main groups (n=48 per group): Experimental group (BetA), positive control group (goniothalamin), vehicle control group (dimethyl sulfoxide) and control group (without vehicle). Each group was further divided into three equal subgroups according to the treatment length (4, 8 and 12 days). BetA treatment induced the expression of NADPH-d activity in the thymus and spleen without any significant changes in the morphology of the organs. Furthermore, the expression pattern of NADPH-d in BetA-treated animals was significantly increased compared with that in the control animals. NADPH-d expression in the thymus and spleen suggests that NO signaling may be a potential mechanism underlying the BetA-induced immunomodulation in these organs. These findings are of direct clinical relevance and may contribute to the further development of BetA as a therapeutic drug.

The evaluation of crude drugs of natural origin as sources of new effective anticancer agents continues to be important due to the lack of effective anticancer drugs currently used in practice which are generally accompanied with adverse effects at different levels of severity. The aim of this concise review is to gather existing literature on anticancer potential of extracts and compounds isolated from Celastraceae species. This review covers six genera (Maytenus, Tripterygium, Hippocratea, Gymnosporia, Celastrus and Austroplenckia) belonging to this family and their 33 isolates. Studies carried out by using different cell lines have shown remarkable indication of anticancer activity, however, only a restricted number of studies have been reported using in vivo tumor models. Some of the compounds, such as triptolide, celastrol and demethylzeylasteral from T. wilfordii, have been extensively studied on their mechanisms of action due to their potent activity on various cancer cell lines. Such promising lead compounds should generate considerable interest among scientists to improve their therapeutic potential with fewer side effects by molecular modification.

SOX4 is a critical developmental transcription factor in vertebrates and is required for precise differentiation and proliferation in multiple tissues. In addition, SOX4 is overexpressed in many human malignancies, but the exact role of SOX4 in cancer progression is not well understood. Here, we have identified the direct transcriptional targets of SOX4 using a combination of genome-wide localization chromatin immunoprecipitation–chip analysis and transient overexpression followed by expression profiling in a prostate cancer model cell line. We have also used protein-binding microarrays to derive a novel SOX4-specific position-weight matrix and determined that SOX4 binding sites are enriched in SOX4-bound promoter regions. Direct transcriptional targets of SOX4 include several key cellular regulators, such as EGFR, HSP70, Tenascin C, Frizzled-5, Patched-1, and Delta-like 1. We also show that SOX4 targets 23 transcription factors, such as MLL, FOXA1, ZNF281, and NKX3-1. In addition, SOX4 directly regulates expression of three components of the RNA-induced silencing complex, namely Dicer, Argonaute 1, and RNA Helicase A. These data provide new insights into how SOX4 affects developmental signaling pathways and how these changes may influence cancer progression via regulation of gene networks involved in microRNA processing, transcriptional regulation, the TGFβ, Wnt, Hedgehog, and Notch pathways, growth factor signaling, and tumor metastasis. [Cancer Res 2009;69(2):709–17]

Photodynamic therapy (PDT) is a promising treatment that is approved by the US FDA for the treatment of oesophageal and lung cancer as well as for age-related macular degeneration. In this study, using standard tissue culture techniques, the photo cytotoxicity and apoptotic mechanisms of Calphostin C (Cal C), a perylenequinone microbial compound in combination with visible light dose was examined in different tumor cell lines. Our results demonstrated both a time and drug-light dose dependence in Cal-C-PDT induced photo toxicity and apoptotic cell death. The induction of apoptosis by Cal C-PDT was found to transit to necrotic cell death at higher drug and light doses. The detection of apoptosis in irradiated tumor cells was performed using various approaches including cell morphology analysis, flow cytometry [DNA fragmentation and phosphatidylserine (PS) externalization] and biochemical assays (activation of caspases). Time-course analysis of Cal C cellular uptake and distribution showed a rapid increase within the cellular compartments. The activation of caspases and nuclear fragmentation was evidenced at a maximum time point of 3 h after irradiation. By the use of specific caspase substrates, significant activation of caspase-8 and -3 was found. Mitochondrial involvement during Cal C-PDT-induced apoptosis was proven by a rapid reduction of the mitochondrial membrane potential. Furthermore, Cal C-PDT also enhanced FasL expression, which then induced Fas signalling-dependent cell death in NPC and colon cancer cell lines tested. Our results contribute to a deeper understanding of the processes involved in apoptotic cell death following photodynamic treatment with Cal C.

Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.

Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene. (Cancer Res 2006; 66(8): 4011-9)

Background We previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. To cross-validate and extend our initial findings, we sought to compare the expression profiles of these cell lines using an alternative microarray platform. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. RESULTS: While we were able to previously identify only 456 genes affected by ST with the Affymetrix platform, we identified 1927 individual genes with the AB platform. Additional technical replicates increased the number of identified genes to 3478 genes and confirmed the changes in 278 (61%) of our original set of 456 genes. Among the 3200 genes newly identified as affected by SV40 ST, we confirmed 20 by QRTPCR including several components of the Wnt, Notch, and Hedgehog signaling pathways, consistent with SV40 ST activation of these developmental pathways. While inhibitors of Notch activation had no effect on cell survival, cyclopamine had a potent killing effect on cells expressing SV40 ST. CONCLUSIONS: These data show that SV40 ST expression alters cell survival pathways to sensitize cells to the killing effect of Hedgehog pathway inhibitors.

Pathological expression of human ErbB-2 protein, also known as HER-2, is common in many types of cancer. ErbB-2 is a member of the EGF receptor tyrosine kinase family and has been rigorously studied as a signaling molecule on the cell membrane. Here, we report that ErbB-2 is also expressed in the nucleus in cultured cells as well as primary tumor tissues. Nuclear ErbB-2 was found to associate with multiple genomic targets in vivo, including the cyclooxygenase enzyme COX-2 gene promoter. ErbB-2 forms a complex at a specific nucleotide sequence of the COX-2 promoter and is able to stimulate its transcription. This study demonstrates the presence of ErbB-2 in the nucleus and identifies the function of ErbB-2 as a transcriptional regulator.

Epidermal growth factor receptor (EGFR) has been detected in the nucleus of cancer cells and primary tumors for decades. While localized in the nucleus, EGFR functions as a transcriptional regulator resulting in the activation of the cyclin D1 gene. Despite nuclear accumulation of EGFR is linked to increased DNA synthesis and proliferative potential, the pathological significance of nuclear EGFR, however, remains uninvestigated. Furthermore, expression of EGFR has not provided a consistent predictive value for survival of breast cancer patients. Here, we analyzed 130 breast carcinomas via immunohistochemical analyses for the levels of nuclear and non-nuclear EGFR. We found 37.7% of the cohort immunostained positively for nuclear EGFR and 6.9% with high levels of expression. Importantly, Kaplan-Meier survival analysis and log-rank test revealed a significant inverse correlation between high nuclear EGFR and overall survival (P = 0.009). Expression of nuclear EGFR correlated positively with increased levels of cyclin D1 and Ki-67, both are indicators for cell proliferation. In contrast, expression of non-nuclear EGFR did not significantly correlate with those of cyclin D1 and Ki-67 or the overall survival rate. In addition, we analyzed 37 oral squamous carcinomas for EGFR expression and found 24.3% of the cases to contain moderate/high levels of nuclear EGFR. Taken together, our findings indicate pathological significance of nuclear EGFR and may have important clinical implication.