Exosome to Promote Cancer Progression via Its Bioactive Cargoes.

Exosomes are nanosized, organelle-like membranous vesicles secreted from various cell types, including normal cells and cancer cells. Exosomes contain abundant bioactive molecules, including nucleic acids, lipids, and proteins and dynamically participate in intercellular communications. By shuttling the functional molecules into the recipient cells, exosomes secreted by cancerous cells can alter the cellular environment to favor tumor growth and metastasis. In this review, we focus on exosomes to promote cancer progression via their various bioactive cargoes through different mechanisms/pathways. By recognizing these pathways, we can design efficient therapeutic strategies to control cancer progression.

Age-related transcriptome changes in melanoma patients with tumor-positive sentinel lymph nodes.

Age is an important factor for determining the outcome of melanoma patients. Sentinel lymph node (SLN) status is also a strong predictor of survival for melanoma. Paradoxically, older melanoma patients have a lower incidence of SLN metastasis but a higher mortality rate when compared with their younger counterparts. The mechanisms that underlie this phenomenon remain unknown. This study uses three independent datasets of RNA samples from patients with melanoma metastatic to the SLN to identify age-related transcriptome changes in SLNs and their association with outcome. Microarray was applied to the first dataset of 97 melanoma patients. NanoString was performed in the second dataset to identify the specific immune genes and pathways that are associated with recurrence in younger versus older patients. qRT-PCR analysis was used in the third dataset of 36 samples to validate the differentially expressed genes (DEGs) from microarray and NanoString. These analyses show that FOS, NR4A, and ITGB1 genes were significantly higher in older melanoma patients with positive SLNs. IRAK3- and Wnt10b-related genes are the major pathways associated with recurrent melanoma in younger and older patients with tumor-positive SLNs, respectively. This study aims to elucidate age-related differences in SLNs in the presence of nodal metastasis.

Comparative gene expression analysis in melanocytes driven by tumor cell-derived exosomes.

Abundant with organelle-like membranous structures, the tumor microenvironment is composed of cancer cells that secrete exosomes. Studies have shown that these secreted exosomes transport RNA and active molecules to other cells to reshape the tumor microenvironment and promote tumor growth. In fact, we found that exosomes derived from melanoma cells drive pre-malignant transition in primary melanocytes. However, there is little available in the scientific literature on how exosomes modulate melanocytes in the microenvironment to optimize conditions for tumor progression and metastasis. We therefore focused this current study on identifying these conditions genetically. Through RNA sequencing, we analyzed gene expression levels of melanocytes driven by exosomes derived from melanoma and lung cancer cells compared with those without exosome controls. Significant differences were found in gene expression patterns of melanocytes driven by exosomes derived from melanoma and lung cancer cells. In the melanocytes responding to exosomes derived from melanoma cells, genes of lipopolysaccharide and regulation of leukocyte chemotaxis were predominant. In the melanocytes responding to exosomes derived from lung cancer cells, genes of DNA replication and mitotic nuclear division played an important role. These results provide further mechanistic understanding of tumor progression promoted by tumor-derived exosomes. This will also help identify potential therapeutic targets for melanoma progression.

Differential expression of ABCB5 in BRAF inhibitor-resistant melanoma cell lines.

BACKGROUND: More than 50% of metastatic melanoma patients have a specific mutation in the serine/threonine kinase BRAF. This results in constitutive activation of the RAS-RAF-MEK-ERK-MAP kinase pathway, which causes uncontrolled cell growth. Vemurafenib (PLX4032) is an oral chemotherapeutic agent that targets the specific mutation V600E in the BRAF protein. Initial response rates are high in patients with BRAF mutant melanoma treated with a BRAF inhibitor such as vemurafenib, but resistance nearly always develops and disease progression ensues. There are several different mechanisms by which melanoma develops BRAF inhibitor resistance. One potential component of resistance is increased drug efflux. Overexpressed ABCB5 (ATP-binding cassette transporter, subfamily B, member 5) has been shown to efflux anti-cancer drugs from cancer cells. The purpose of this study is to determine whether ABCB5 is highly expressed in BRAF inhibitor-resistant melanoma cells and to evaluate whether ABCB5 is involved in the development of resistance to BRAF inhibitors in cutaneous melanoma. METHODS: We established three BRAF inhibitor-resistant melanoma cell lines with BRAF mutation. The expression level of ABCB5 in PLX-resistant cell lines was checked by real-time PCR and Western blot analysis. SK-MEL-2 melanoma cells with wild-type BRAF were used for comparison. The association of different levels of ABCB5 with the changes of ERK, p-ERK, Akt and p-Akt was also assessed by Western blotting. Re-sensitization of melanoma cells to PLX was tested by p-ERK inhibitor PD58059 and ABCB5 knockdown by ABCB5 siRNA, respectively. RESULTS: We showed that ABCB5 was overexpressed in SK-MEL-28PLXr and A2058PLXr cells but not in A375PLXr cells. ABCB5 overexpression is associated with activation of p-ERK status but not Akt. Inhibition of p-ERK re-sensitized SK-MEL-28PLXr and A2058PLXr cells to PLX treatment, but knockdown of ABCB5 did not re-sensitize A2058 PLXr and SK-MEL-28 PLXr cells to PLX treatment. CONCLUSION: These results confirm that, even though ABCB5 was overexpressed in SK-MEL-28 and A2058 melanoma cells that develop resistance to BRAF inhibitors, ABCB5 may not be a major targetable contributor to BRAF resistance. p-ERK inhibition may play important roles in BRAF resistance in these two melanoma cell lines.

Unique Genes in Tumor-Positive Sentinel Lymph Nodes Associated with Nonsentinel Lymph Node Metastases in Melanoma.

BACKGROUND: Current risk assessment tools to estimate the risk of nonsentinel lymph node metastases after completion lymphadenectomy for a positive sentinel lymph node (SLN) biopsy in cutaneous melanoma are based on clinical and pathologic factors. We identified a novel genetic signature that can predict non-SLN metastases in patients with cutaneous melanoma staged with a SLN biopsy. METHODS: RNA was collected for tumor-positive SLNs in patients staged by SLN biopsy for cutaneous melanoma. All patients with a tumor-positive SLN biopsy underwent completion lymphadenectomy. A 1:10 case:control series of positive and negative non-SLN patients was analyzed by microarray and quantitative RT-PCR. Candidate differentially expressed genes were validated in a 1:3 case:control separate cohort of positive and negative non-SLN patients. RESULTS: The 1:10 case:control discovery set consisted of 7 positive non-SLN cases matched to 70 negative non-SLN controls. The cases and controls were similar with regards to important clinicopathologic factors, such as gender, primary tumor site, age, ulceration, and thickness. Microarray and RT-PCR identified six potential differentially expressed genes for validation. In the 40-patient separate validation set, 10 positive non-SLN patients were matched to 30 negative non-SLN controls based on gender, ulceration, age, and thickness. Five of the six genes were differentially expressed. The five gene panel identified patients at low (7.1%) and high risk (66.7%) for non-SLN metastases. CONCLUSIONS: A novel, non-SLN gene score based on differential expressed genes in a tumor-positive SLN can identify patients at high and low risk for non-SLN metastases.

Sentinel Lymph Node Genes to Predict Prognosis in Node-Positive Melanoma Patients.

PURPOSE: Melanoma patients with a single microscopically-positive sentinel lymph node (SLN) are classified as stage III and are often advised to undergo expensive and substantially toxic adjuvant therapy. However, the 5-year survival rate for these patients, with or without adjuvant therapy, varies from 14 to 85 %, representing a heterogeneous biological population with a variable prognosis. We aimed to identify an SLN gene signature to aid in risk stratification of patients with tumor-positive SLNs. METHODS: Microarray experiments were performed to screen SLN genes in recurrence (N = 39) versus non-recurrence (N = 58) groups in the training dataset. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) assay was applied to confirm the expression of selected SLN genes, which were further verified using an independent validation cohort (N = 30). Area under the receiver operating characteristic curve (AUC) was calculated to evaluate prognostic accuracy of the selected SLN gene panel, and the prognostic value of our SLN gene signature was also compared with the current American Joint Committee on Cancer (AJCC) staging system. RESULTS: We identified two SLN genes (PIGR and TFAP2A) that provided high prognostic accuracy in SLN-positive melanoma patients (AUC = 0.864). These two SLN genes, along with clinicopathological features, can differentiate the high- and low-risk groups in node-positive melanoma patients in this cohort. CONCLUSION: The two SLN genes, when combined with clinicopathological features, may offer a new tool for personalized patient risk assessment.

Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment.

The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma.

Hematite nanoplates: Controllable synthesis, gas sensing, photocatalytic and magnetic properties.

Uniform hematite (α-Fe2O3) nanoplates exposing {001} plane as basal planes have been prepared by a facile solvothermal method under the assistance of sodium acetate. The morphological evolution of the nanoplates was studied by adjusting the reaction parameters including the solvent and the amount of sodium acetate. The results indicated that both the adequate nucleation/growth rate and selective adsorption of alcohol molecules and acetate anions contribute to the formation of the plate-like morphology. In addition, the size of the nanoplates can be adjusted from ca. 180nm to 740nm by changing the reaction parameters. Three nanoplate samples with different size were selected to investigate the gas sensing performance, photocatalytic and magnetic properties. As gas sensing materials, all the α-Fe2O3 nanoplates exhibited high gas sensitivity and stability toward n-butanol. When applied as photocatalyst, the α-Fe2O3 nanoplates show high photodegradation efficiency towards RhB. Both the gas sensing performance and the photocatalytic property of the products exhibit obvious size-dependent effect. Magnetic measurements reveal that the plate-like α-Fe2O3 particles possess good room temperature magnetic properties.

Identifying mRNA, microRNA and protein profiles of melanoma exosomes.

BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

Adenovirus-mediated expression of mutated forkhead human transcription like-1 suppresses tumor growth in a mouse melanoma xenograft model.

Melanoma is generally resistant to chemotherapy, which may be related to defects in death receptor signaling and to defects in induction of apoptosis. Forkhead family transcription factors induce the expression of death receptor ligands such as Fas ligand (Fas-L) resulting in apoptosis. We therefore investigated whether a triple mutant form of forkhead transcription factor FKHRL1 (FKHRL1/TM) can enhance Fas-L mediated-apoptosis in melanoma cells. Two melanoma cells A2058 or DM6 were tested for their sensitivity to agonistic anti-Fas antibody (CH-11); adenovirus expressing FKHRL1/TM (Ad-FKHRL1/TM) was assessed for its capability to induce activation of the caspase pathway; the role of Fas-L in the Ad-FKHRL1/TM mediated-cell death was also assessed in vitro. Ad-FKHRL1/TM antitumor activity in vivo was also evaluated in a mouse melanoma xenograft model. We found that DM6 melanoma cells were more resistant to Fas/Fas-L-mediated apoptosis induced by agonistic anti-Fas antibody than A2058 melanoma cells. Ectopic expression of FKHRL1/TM in melanoma cells upregulated Fas-L expression, decreased procaspase-8 levels, and significantly increased Fas/FasL-mediated cell death in both cells lines; this induced cell death was partially blocked by a Fas/Fas-L antagonist. Importantly, Ad-FKHRL1/TM treatment of subcutaneous melanoma xenografts in mice resulted in approximately 70% decrease in tumor size compared with controls. These data indicate that overexpression of FKHRL1/TM can induce the Fas-L pathway in melanoma cells. Ad-FKHRL1/TM therefore might represent a promising vector for melanoma treatment.

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK.

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.

Adenovirus-mediated expression of truncated E2F-1 suppresses tumor growth in vitro and in vivo.

BACKGROUND: Adenovirus (Ad)-mediated E2F-1 gene transfer induces apoptosis in cancer cells in vitro and in vivo, but clinical application of E2F-1 in cancer gene therapy remains controversial because of the oncogenic potential of E2F-1. This barrier can be circumvented by using the truncated form of the E2F-1 gene (E2Ftr) (amino acids 1 through 375), which lacks the E2F-1 transactivation domain and cell cycle-promoting effects. METHODS: The authors constructed 3 adenoviral vectors that expressed E2Ftr under regulation of the tetracycline (Tet)-off system (AdTet-E2Ftr1, AdTet-E2Ftr2, and AdTet-E2Ftr3). These vectors were compared for E2Ftr expression and apoptosis induction in cancer cells and normal cells. E2Ftr antitumor activity in vivo also was assessed in a melanoma xenograft model. RESULTS: One of the 3 vectors, AdTet-E2Ftr3, had the highest E2Ftr protein expression levels, which were correlated with the greatest induction of apoptosis and inhibition of cancer cell growth. E2Ftr induced apoptosis in a variety of cancer cell lines independent of p53 status with little cytotoxicity in normal cell lines. In a mouse melanoma xenograft model, AdTet-E2Ftr3 exhibited an approximately 80% decrease in tumor size compared with controls in vivo. CONCLUSIONS: The current results indicated that AdTet-E2Ftr3 is a novel anticancer agent that has significant therapeutic activity in vitro and in vivo.

Developing adenoviral vectors encoding therapeutic genes toxic to host cells: comparing binary and single-inducible vectors expressing truncated E2F-1.

Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation.

Chemosensitization of tumor cells: inactivation of nuclear factor-kappa B associated with chemosensitivity in melanoma cells after combination treatment with E2F-1 and doxorubicin.

Combination chemotherapy has been shown to be more effective than single-agent therapy for many types of cancer, but both are known to induce drug resistance in cancer cells. Two major culprits in the development of this drug resistance are nuclear factor-kappaB (NF-kappaB) and the multidrug resistance (MDR) gene. For this reason, chemogene therapy is emerging as a viable alternative to conventional chemotherapy combinations. We have shown that transduction of the E2F-1 gene in melanoma cells markedly increases cell sensitivity to doxorubicin, thereby producing a synergistic effect on melanoma cell apoptosis. Our microarray results show that the NF-kappaB pathway and related genes undergo significant changes after the combined treatment of E2F-1 and doxorubicin. In fact, inactivation of NF-kappaB is associated with melanoma cell apoptosis induced by E2F-1 and doxorubicin, providing a link between the NF-kappaB signaling pathway and the chemosensitivity of melanoma cells after this treatment.

Adenovirus E1B55K region is required to enhance cyclin E expression for efficient viral DNA replication.

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G(0) phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G(0) phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

Induction of apoptosis signal-regulating Kinase 1 by E2F-1 may not be essential for E2F-1-mediated apoptosis in melanoma cells.

OBJECTIVES: In the present study, we investigate the role of apoptosis signal-regulating kinase 1 (ASK1) mitogen-activated protein (MAP) kinase signal pathways in E2F-1-mediated apoptosis. METHODS: A gene expression profile in response to E2F-1 overexpression was performed by cDNA microarray analysis and confirmed by real-time reverse-transcription polymerase chain reaction. Kinase activities were assayed by Western blot analysis or kinase assay. Apoptosis was assessed by morphologic inspection and flow-cytometric analysis. Cytotoxicity was monitored by MTT assay. RESULTS: E2F-1 upregulated the expression of ASK1 8-fold compared to the Ad-LacZ-infected control in SK-MEL-2 melanoma cells, which was confirmed by reverse-transcription polymerase chain reaction. Sequence analysis showed that there are 2 putative E2F-1 DNA binding sites in the ASK1 promoter region. Truncated E2F-1 protein, which lacks the transactivation domain, failed to upregulate ASK1, suggesting that ASK1 was regulated at the transcriptional level by E2F-1. E2F-1 overexpression resulted in the transient activation of c-Jun N-terminal kinase (JNK); however, dominant negative mutant ASK1 had no effect on E2F-1 cytotoxicity and JNK activation. p38 was not activated by E2F-1, and inhibition of p38 had no effect on E2F-1-mediated cell death. The ASK1 kinase assay showed that ASK1 activity was not upregulated in response to E2F1 overexpression. The inhibition of ASK1 upstream kinase-AKT can enhance E2F-1-mediated cell death. Moreover, an adenovirus expressing truncated E2F-1 keeps the ability of inducing apoptosis in melanoma cells. CONCLUSIONS: ASK1 expression is upregulated by E2F-1 at the transcription level, but the upregulation of ASK1 expression by E2F-1 was not coordinated with an increased ASK1 activity. The ASK1-JNK/p38 pathway does not appear to play a crucial role in E2F-1-induced apoptosis.

E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation.

BACKGROUND: PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. METHODS: PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. RESULTS: Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. CONCLUSION: Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy.

Alteration of gene expression in melanoma cells following combined treatment with E2F-1 and doxorubicin.

BACKGROUND: The combination of E2F-1 gene therapy and chemotherapy produces a synergistic effect on melanoma cell apoptosis. However, the molecular mechanisms have not been fully elucidated. The purpose of this study was to identify novel genes or pathways that may play key roles in apoptosis when E2F-1 gene therapy is combined with doxorubicin chemotherapy. MATERIALS AND METHODS: SK-MEL-2 melanoma cells were infected with Ad-E2F-1 alone, Ad-E2F-1 plus doxorubicin, or Ad-LacZ plus doxorubicin. After 16 hours of treatment, the total RNA was extracted from these cells and subjected to microarray analysis. Quantitative real-time PCR was performed to confirm the microarray data. RESULTS: Our results showed that the combination treatment of Ad-E2F-1 and doxorubicin affected the expression of cytokines, transcription factors, as well as genes involved in signal transduction, cell cycle regulation and apoptosis. CONCLUSION: Our findings have identified, for the first time, novel molecular targets and pathways that led to apoptosis in melanoma cells when Ad-E2F-1 was combined with doxorubicin. The molecular information provided here will enhance further mechanistic studies.

[Mechanism of tetra-arsenic tetra-sulfide in inducing apoptosis of acute promyelocytic leukemia cells].

OBJECTIVE: To investigate the mechanism of tetra-arsenic tetra-sulfide (As4S4) in inducing apoptosis of acute promyelocytic leukemia (APL) cells. METHODS: The gene expression patterns in NB4 cells pre- and post-treatment with As4S4 were analyzed by cDNA microarray, and differentially expressed genes related with apoptosis were identified. The mRNA expression levels of these apoptosis related genes in the peripheral blood of APL patients treated with As4S4 pre- and post-remission were examined by RT-PCR. RESULTS: Among the differentially expressed genes in NB4 cells pre- and post-treatment with As4S4, two genes were related with cellular apoptosis, which were Apaf-1 and PNAS-2. Apaf1 ratio between pre- and post- As4S4 in NB4 cells was 2.910, PNAS-2 ratio was 0.420. RT-PCR results showed that the expression ratios of Apaf-1, caspase-9 and PNAS-2 in APL patients pre- and post-remission were 2.31 and 3.21, 0.99 respectively. CONCLUSION: As4S4 induced cellular apoptosis in NB4 cells involves the expression changes of the two genes: the up-regulation of Apaf1 and down-regulation of PNAS-2. The increased expressions of Apaf1 and caspase-9 indicate that As4S4 induced apoptosis in APL cells is through mitochondrial pathway, but not the death-receptor pathway. The role played by PNAS-2 in cellular apoptosis needs to be clarified.

[Study on the role of PML-RARalpha and RARalpha fusion proteins in NB4 cell apoptosis induced by arsenic trisulfide].

In order to study the role of PML-RARalpha fusion gene and its expression product during apoptotic process in NB4 cells induced by arsenic trisulfide (As(2)S(3)), the apoptotic effects of NB4 cells were observed by cell morphology, flow cytometry and DNA electrophoresis. The change of PML-RARalpha fusion gene and its expression product were also assayed by chromosomal G banding, RT-PCR and Western blot. The results showed that arsenic trisulfide induced apoptosis of NB4 cells, during this process, PML-RARalpha fusion gene had no significant changes, but the expression of PML-RARalpha fusion protein and wild-type RARalpha were all reduced. It is concluded that arsenic trisulfide can induce apoptosis of NB4 cells, the degradation of PML-RARalpha fusion protein and wild-type RARalpha may play an important role during apoptotic process.