G361 Cells Research Articles

Dichlorido-platinum(II) complexes with kinetin derivatives as promising cytotoxic agents avoiding resistance of cancer cells: Contrasting results between cisplatin and oxaliplatin analogues

With the aim to study the anticancer potential and compare the influence of different leaving groups, dichlorido and oxalato (ox) platinum(II) complexes involving di- and tri-substituted derivatives of a plant hormone kinetin (N6-furfuryladenine; Ln) were prepared: cis-[PtCl2(L1)2]·H2O (1), trans-[PtCl2(L1)2] (1a), cis-[PtCl2(L2)2]·0.75H2O (2), and [Pt(L1)2(ox)] (3), [Pt(L2)2(ox)] (4), with 2-chloro-N6-furfuryl-9-isopropyladenine (L1) and 2-chloro-N6-(5-methylfurfuryl)-9-isopropyladenine (L2). The complexes were structurally characterized by elemental and thermal analyses, FT-IR and 1H, 13C, 15N and 195Pt NMR spectroscopy, ESI+ mass spectrometry, conductivity measurements as well as single crystal X-ray diffraction (for 1). The Ln carrier ligands act as monodentate N-donors coordinated to the platinum(II) centre, which reveals a slightly distorted square-planar geometry. All the prepared complexes were screened for their in vitro cytotoxicity against breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines. This preliminary study identified only the cis-dichlorido-Pt(II) complexes 1, 2 as cytotoxic agents. These two complexes were thus further evaluated for in vitro cytotoxicity against malignant melanoma (G-361), cervix epitheloid carcinoma (HeLa), cisplatin sensitive (A2780) and resistant (A2780R) ovarian carcinoma human cancer cell lines. Most importantly, the obtained results showed that the cis-[PtCl2(Ln)2]·xH2O complexes were able to circumvent cisplatin resistance in A2780R cells while additionally being significantly more cytotoxic against A2780 than cisplatin, as demonstrated by the resistance factors IC50(A2780R)/IC50(A2780) equal to 0.87 for 1; 0.48 for 2 as compared to 2.25 for cisplatin. The complexes were furthermore comparably active against HOS, MCF7, G-361 cells as cisplatin. In order to address the contrasting cytotoxic results of the cisplatin (1, 2) and oxaliplatin (3, 4) analogues, their possible fate in the physiological environment, focusing on the hydrolytic stability and interactions with glutathione and 5′-guanosine monophosphate, was studied by means of 1H NMR spectroscopy.

C-MYC and C-FOS expression changes and cellular aspects of the photodynamic reaction with photosensitizers TMPyP and ClAlPcS2

Photodynamic therapy (PDT) is based on the tumor-selective accumulation of photosensitizer followed by irradiation with light of an appropriate wavelength. After irradiation and in the presence of oxygen, photosensitizer induces cellular damage. The aim of this study was to evaluate effects of two photosensitizers TMPyP and ClAlPcS2 on cell lines to obtain better insight into their mechanisms of action. We determined cell viability, reactive oxygen species (ROS) generation and changes in expression levels of two important early response genes, C-MYC and C-FOS, on tumor MCF7 (human breast adenocarcinoma) and G361 (human melanoma) cell lines and non-tumor BJ cell line (human fibroblast) after photodynamic reaction with TMPyP and ClAlPcS2 as photosensitizers. In addition TMPyP and ClAlPcS2 cellular uptake and clearance and antioxidant capacity of the mentioned cell lines were investigated. We found appropriate therapeutic doses and confirmed that both tested photosensitizers are photodynamically efficient in treatment used cells in vitro. TMPyP is more efficient; it had higher ROS production and toxicity after irradiation by intermediate therapeutic doses than ClAlPcS2. We revealed that both TMPyP and ClAlPcS2-PDT increased C-FOS expression on tumor cell lines (G361 and MCF7), but not on non-tumor BJ cell line. Conversely, both TMPyP and ClAlPcS2-PDT decreased C-MYC expression on non-tumor BJ cell line but not on tumor cell lines. As first we tested these photosensitizers in such extent and we believe that it can help to better understand mechanisms of PDT and increase its efficiency and applicability.

Abstract 1348: Suppressor of cytokine signaling (SOCS)-1 suppresses a proliferation of malignant melanoma cells via the suppression of JAK/STAT and the activation of p53 signaling pathways

Abstract BACKGROUND : Melanoma is the most aggressive form of skin cancer, responsible for the majority of skin cancer related deaths. JAK-STAT signal is one of the main pathways involved in the tumor growth in melanoma, because JAK inhibitor has been reported to induce apoptosis in melanoma cells. The suppressor of cytokine signaling (SOCS) family proteins are important negative regulator of proinflammatory cytokine signals by suppressing JAK-STAT signal pathway via inhibiting JAK activity. The aim of this study was to evaluate the role of SOCS-1 in the proliferation of melanoma through regulating cytokine signaling pathway and to clarify the mechanisms of its antitumor effect in vitro and in vivo. MATERIAL AND METHODS : Adenovirus vectors encoding SOCS1 gene was used to overexpress SOCS1 in three melanoma cell lines (G361, SK-MEL5, and SK-MEL28). The status of activated kinases and downstream proteins were determined by Western blot analysis. Anti-proliferative effects in melanoma cells were assessed by MTT assay. Apoptosis in melanoma cells was assessed by Caspase 3 activity and TUNEL staining. ICR nu/nu mice were used for subcutaneous xenograft experiment. RESULTS : High levels of phosphorylated STAT3 in G361 and SK-MEL5, but not in SK-MEL28. Overexpression of SOCS1 significantly reduced cell proliferation and induced apoptosis in G361 and SK-MEL5. Furthermore, in G361 and SK-MEL5 xenograft model, intratumoral injection of AdSOCS1 vector significantly reduced tumor volumes compared to AdLacZ-injected mice. Western blot and immunohistochemical analysis indicated that SOCS1 was overexpressed and induced apoptosis in the tissues from AdSOCS1-injected mice. The anti-proliferative effect of SOCS1 correlated with decreased levels of the activation of STAT3 and increased levels of p53. Furthermore the G0/G1 cell population was increased by overexpression of SOCS1 in G361. On the other hand, in SK-MEL28, SOCS1 delivery didn't reduce cell proliferation so markedly. CONCLUSIONS : We showed that overexpression of SOCS-1 suppresses a proliferation of G361 and SK-MEL5 malignant melanoma cells via the suppression of JAK/STAT and the activation of p53 signaling pathways. Overexpression of SOCS-1 resulted in the cell cycle arrest at G1/S phase. Furthermore, we firstly demonstrated that overexpression of SOCS-1 induced apoptosis in melanoma cells in vitro and in vivo. Therefore, low levels of phosphorylated STAT3 as well as a mutation of p53 in SK-MEL28 may be the reason why SOCS1 didn't reduce cell proliferation so markedly in SK-MEL28. We hope that these findings may lead to the successful clinical application of SOCS1 for melanoma treatment. Citation Format: Naoko Tagami, Satoshi Serada, Minoru Fujimoto, Atsushi Tanemura, Ichiro Katayama, Tetsuji Naka. Suppressor of cytokine signaling (SOCS)-1 suppresses a proliferation of malignant melanoma cells via the suppression of JAK/STAT and the activation of p53 signaling pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1348. doi:10.1158/1538-7445.AM2014-1348

Effect of telomerase inhibition on preclinical models of malignant rhabdoid tumor

Novel treatment approaches are desperately needed for malignant rhabdoid tumor (MRT). Telomerase is an attractive therapeutic target because it is specific to cancer and critical for cancer cell immortality. We evaluated the effect of the telomerase inhibitor imetelstat in preclinical models of MRT. Three MRT cell lines, BT-12, G401, and RT-peri, were treated with the telomerase inhibitor imetelstat. The effects of imetelstat on telomere length, DNA damage response, and cell proliferation were assessed. The efficacy of imetelstat invivo was evaluated in subcutaneous xenografts derived from each of the cell lines. Treatment with imetelstat resulted in inhibition of telomerase activity, marked telomere shortening, and activation of the DNA damage response pathway, as measured by formation of γ-H2AX nuclear foci, phosphorylation of ATM, and phosphorylation of TP53. Imetelstat-treated G401 cells underwent complete growth arrest after 16 passages. The other two cell lines exhibited growth inhibition. Imetelstat resulted in 40-50% growth inhibition compared to placebo-treated controls in all three xenograft models. The activity of imetelstat as a single agent suggests that further studies of telomerase inhibitors in combination with other agents may be warranted.

Synthesis, characterization and in vitro antitumor activity of platinum(II) oxalato complexes involving 7-azaindole derivatives as coligands.

The platinum(II) oxalato complexes [Pt(ox)(naza)2] (1–3) were synthesized and characterized by elemental analysis (C, H, N), multinuclear NMR spectroscopy (1H, 13C, 15N, 195Pt) and electrospray ionization mass spectrometry (ESI-MS); naza = 4-chloro-7-azaindole (4Claza; 1), 3-bromo-7-azaindole (3Braza; 2) or 4-bromo-7-azaindole (4Braza; 3). The prepared substances were screened for their in vitro antitumor activity on the osteosarcoma (HOS) and breast adenocarcinoma (MCF7) human cancer cell lines, where 2 showed moderate antitumor effect (IC50 = 27.5 μM, and 18.3 μM, respectively). The complex 2 was further tested on a panel of six others human cancer cell lines, including the malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate adenocarcinoma (LNCaP). This substance was found to be moderate antitumor effective against G361 (IC50 = 17.3 μM), HeLa (IC50 = 31.8 μM) and A2780 (IC50 = 19.2 μM) cell lines. The complex 2 was also studied by NMR for its solution stability and by ESI-MS experiments for its ability to interact with biomolecules, such as cysteine, glutathione or guanosine 5'-monophosphate.

Combined inhibition of NF-κB and Bcl-2 triggers synergistic reduction of viability and induces apoptosis in melanoma cells.

Constitutive activation of nuclear factor κB (NF-κB) characterizes melanoma cells. To explore the molecular mechanism of melanoma cell survival by constitutive NF-κB activation, we used the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), which directly binds to NF-κB. DHMEQ abrogated constitutive NF-κB activity, which included RelA (p65)/p50 in melanoma cell lines G361 and HMV-II; however, the reduction of the viability was marginal. Expression of c-FLIP was not observed in the melanoma cell lines tested, and DHMEQ could not repress the expression of the Bcl-2 family proteins Bcl-2 and Bcl-xL. Concomitant treatment with DHMEQ and the inhibitor of antiapoptotic Bcl-2 family proteins, GX15-070, triggered synergistic reduction of the viability and induced apoptosis of G361 cells. These results indicate that abrogation of the NF-κB pathway alone is not sufficient to suppress the survival of melanoma cells. The NF-κB and the antiapoptotic Bcl-2 pathways cooperatively support the survival, and the dual targeting triggers synergistic reduction of the viability and induces apoptosis of melanoma cells.

Novel downstream molecular targets of SIRT1 in melanoma: a quantitative proteomics approach.

Melanoma is one of the most lethal forms of skin cancer and its incidence is continuing to rise in the United States. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. SIRT1, a NAD(+)-dependent class III histone deacetylase, has been implicated in a variety of physiological processes and pathological conditions. We recently demonstrated that SIRT1 is upregulated in melanoma and its inhibition by a small-molecule, tenovin-1, inhibits cell proliferation and clonogenic survival of melanoma cells, possibly via activating p53. Here, we employed a gel free quantitative proteomics approach to identify the downstream effectors and targets of SIRT1 in melanoma. The human malignant melanoma, G361 cells were treated with tenovin-1 followed by protein extraction, in liquid trypsin digestion, and peptide analyses using nanoLC-MS/MS. A total of 1091 proteins were identified, of which 20 proteins showed significant differential expression with 95% confidence interval. These proteins were subjected to gene ontology and Ingenuity Pathway Analysis (IPA) to obtain the information regarding their biological and molecular functions. Real-Time qRT-PCR validation showed that five of these (PSAP, MYO1B, MOCOS, HIS1H4A and BUB3) were differentially expressed at mRNA levels. Based on their important role in cell cycle regulation, we selected to focus on BUB family proteins (BUB3, as well as BUB1 and BUBR1) for subsequent validation. The qRT-PCR and immunoblot analyses showed that tenovin-1 inhibition of SIRT1 resulted in a downregulation of BUB3, BUB1 and BUBR1 in multiple melanoma cell lines. Since tenovin-1 is an inhibitor of both SIRT1 and SIRT2, we employed lentivirus mediated silencing of SIRT1 and SIRT2 in G361 cells to determine if the observed effects on BUB family proteins are due to SIRT1- or SIRT2- inhibition. We found that only SIRT1 inhibition resulted in a decrease in BUB3, BUB1 and BUBR1. Our study identified the mitotic checkpoint regulator BUB family proteins as novel downstream targets of SIRT1. However, further validation is needed in appropriate models to confirm our findings and expand on our observations.

DNA aptamer raised against advanced glycation end products inhibits melanoma growth in nude mice

Epidemiological studies have suggested that diabetes is associated with an increased risk of cancer. However, the underlying molecular mechanism remains unclear. We investigated here whether DNA aptamer directed against advanced glycation end products (AGE-aptamer) inhibited melanoma growth in nude mice. G361 melanoma cells were injected intradermally into the upper flank of athymic nude mice. Mice received continuous intraperitoneal infusion (0.136 μg/day) of either AGE-aptamer (n=9) or Control-aptamer (n=8) by an osmotic mini pump. Tumor volume was measured at 4-day interval, and G361 melanoma was excised at day 43 after the aptamer treatment. We further examined the effects of AGE-aptamer on proliferation of AGE-exposed endothelial cells and G361 cells. AGE-aptamer significantly inhibited the in vivo-tumor growth of G361 melanoma. Immunohistochemical and western blotting analyses of G361 melanoma revealed that AGE-aptamer decreased expression levels of proliferating nuclear antigen, CD31 and Mac-3, markers of endothelial cells and macrophages, respectively. AGE-aptamer significantly decreased the number of tumor-associated vessels. AGE, receptor for AGE (RAGE) and vascular endothelial growth factor levels were also reduced in AGE-aptamer-treated G361 melanoma. AGE-aptamer inhibited the AGE-induced proliferation and tube formation of endothelial cells as well as the growth of G361 cells in vitro. The present findings suggest that AGE-aptamer could inhibit the AGE–RAGE axis in G361 melanoma and resultantly suppress the tumor growth in nude mice by blocking the angiogenesis. AGE-aptamer might be a novel therapeutic strategy for preventing the progression of malignant melanoma in diabetes.

Frequent Overexpression of HMGA2 in Human Atypical Teratoid/Rhabdoid Tumor and Its Correlation with let-7a3/let-7b miRNA

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive pediatric malignancies characterized by biallelic inactivation of the SMARCB1 tumor suppressor gene. We searched for novel genomic aberrations by investigating the copy number and expression alterations of let-7a3/let-7b microRNA (miRNA) and correlated these with expression of high-mobility group AT-hook 2 (HMGA2) oncoprotein, a target of let-7 miRNA family, in 18 AT/RT samples to elucidate potential roles of HMGA2 in the pathogenesis of AT/RT. Genomic aberrations, let-7a3/let-7b miRNA and HMGA2 expression in AT/RT tissues were identified using quantitative PCR, reverse transcription PCR (RT-PCR), and immunohistochemistry. The impact of let-7b miRNA on HMGA2 expression and the malignant potential of human rhabdoid tumor cell G401 (SMARCB1(-/-)) were investigated by antisense inhibition and ectopic overexpression studies. The copy number of let-7a3/let-7b miRNA was substantially decreased in 4 of 11 AT/RT samples. A significantly inverse correlation between let-7a3/let-7b miRNA expression and HMGA2 mRNA expression was observed in AT/RT tissues (R = -0.34; P < 0.05). Immunohistochemistry analysis demonstrated that HMGA2 was highly overexpressed in 83.3% (15 of 18) of AT/RT tissues. Restoration of let-7 miRNA or knockdown of HMGA2 expression significantly suppressed proliferation and colony formation, and almost abolished the invasive potential of G401 cells. Reduction of let-7a3/let-7b miRNA may be one of mechanisms leading to overexpression of HMGA2 in AT/RT tissues. HMGA2 oncoprotein plays critical roles in the pathogenesis of AT/RT development; and reconstitution of let-7 miRNA or knockdown of HMGA2 oncoprotein may provide a novel therapeutic strategy for the treatment of patients with AT/RT.

Pheophorbide a-mediated photodynamic therapy induces autophagy and apoptosis via the activation of MAPKs in human skin cancer cells

Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitizer that can induce significant antitumor effects in several types of tumor cells. The present study investigated the mechanism of Pa-mediated photodynamic therapy (Pa-PDT) in the human skin cancer cell lines A431 and G361. PDT significantly inhibited the cell growth in a Pa-concentration-dependent manner. We observed increased expression of Beclin-1, LC3B and ATG5, which are markers of autophagy, after PDT treatment in A431 cells but not in G361 cells. In G361 cells, Pa-PDT strongly induced PARP cleavage and subsequent apoptosis, which was confirmed using Annexin V/Propidium iodide double staining. Pa-PDT predominantly exhibited its antitumor effects via activation of ERK1/2 and p38 in A431 and G361 cells, respectively. An in vivo study using the CAM xenograft model demonstrated that Pa-PDT strongly induced autophagy and apoptosis in A431-transplanted tumors and/or apoptosis in G361-transplanted tumors. These results may provide a basis for understanding the underlying mechanisms of Pa-PDT and for developing Pa-PDT as a therapy for skin cancer.

Berberine inhibits Wilms’ tumor cell progression through upregulation of Wilms’ tumor gene on the X chromosome

Wilms' tumor is a type of kidney cancer that affects young children. Although a number of Wilms' tumor samples have been collected through international trials, the mechanisms underlying its progression remain challenging to determine. Extensive studies have identified somatic mutations at several loci in Wilms' tumorigenesis, including WT1, catenin, Wilms' tumor gene on the X chromosome (WTX) and TP53. Berberine is a benzylisoquinoline alkaloid extracted from numerous types of medicinal plants and has been extensively used as a Chinese traditional medicine. Recently, berberine has been demonstrated to possess antitumoral activities. AMP-activated protein kinase (AMPK) is suggested to be one of the various cellular targets of berberine, which regulates tumor progression and metastasis. However, the specific involvement of berberine‑induced AMPK activation and its effects on the proliferation potential of Wilms' tumor cells remains unknown. The present study investigated the berberine‑induced activation of AMPK and its effects on G401 Wilms' tumor cell proliferation. The results demonstrated that berberine inhibited growth and decreased the expression of cell‑cycle regulators in these cells. At the molecular level, berberine treatment led to a significant increase of WTX expression and G401 cells were protected against berberine‑induced growth inhibition by small interfering RNA against WTX. In conclusion, these results suggest a novel mechanism that may contribute to the antineoplastic effects of berberine which was also demonstrated by recent population studies; however, further studies are required to investigate the potential therapeutic use of berberine in patients with Wilms' tumor.

Application of EPR spectroscopy to examination of free radicals in melanins from A-375 and G-361 human melanoma malignum cells

Melanins are polymorphous and multifunctional biopolymers with a relatively high concentration of free radicals. EPR spectroscopy was used to study o-semiquinone free radicals in model eumelanins synthesized from 3,4-dihydroxyphenylalanine (DOPA) and tyrosine in the presence of tyrosinase, and melanins isolated from A-375 and G-361 human melanoma malignum cells exposed to two compounds: 5,7-dimethoxycoumarin (DMC) and valproic acid (VPA). Changes were determined in the concentrations of free radicals in the individual melanins from tumour cells treated with DMC and VPA. A strong decrease in the concentrations of free radicals characterizes melanins isolated from tumour cells treated together with DMC and VPA. Slow spin-lattice relaxation processes were noted in the melanins tested with homogeneous broadened EPR spectra. The EPR technique may be useful not only for the elucidation of free radicals in melanins from A-375 and G-361 cells treated with VPA and DMC but it could also be applied to establish the relationship between melanin type and the malignancy of melanoma malignum.

Protective effect of AVS073, a polyherbal formula, against UVA-induced melanogenesis through a redox mechanism involving glutathione-related antioxidant defense

BackgroundAyurved Siriraj Brand Wattana formula (AVS073), a Thai herbal formula, has traditionally been used for health promotion and prevention of age-related problems. Ultraviolet A (UVA) is recognized to play a vital role in stimulation of melanin synthesis responsible for abnormal skin pigmentation possibly mediated by photooxidative stress. We thus aimed to study the inhibitory effect of AVS073 extracts on UVA-induced melanogenesis via a redox mechanism involving glutathione (GSH) synthesis and glutathione S-transferase (GST) using human melanoma (G361) cell culture.MethodsThe standardization of AVS073 extracts was carried out by TLC and UHPLC to obtain fingerprinting profiles of the formula, which identified several phenolic compounds including gallic acid (GA) in the formula. Antimelanogenic actions of AVS073 (up to 60 μg/ml) and GA (up to 10 μg/ml) were investigated by measuring tyrosinase activity and mRNA as well as melanin level in G361 cells irradiated with UVA. Moreover, antioxidant actions of the herbal formula and GA were determined by evaluating oxidant formation and modulation of GSH-related antioxidant defenses including GSH content, GST activity and mRNA level of γ-glutamate cysteine ligase catalytic (γ-GCLC) and modifier (γ-GCLM) subunit and GST.ResultsAVS073 extracts and GA, used as a reference compound, suppressed UVA-augmented tyrosinase activity and mRNA and melanin formation. In addition, pretreatment with AVS073 and GA was able to inhibit cellular oxidative stress, GSH depletion, GST inactivation and downregulation of γ-GCLC, γ-GCLM and GST mRNA in G361 cells exposed to UVA radiation.ConclusionsAVS073 formula exerted antimelanogenic effects possibly through improving the redox state by upregulation of GSH and GST. Moreover, pharmacological activity of the polyherbal formula would be attributed to combined action of different phenolic compounds present in the formula.

Exposure to valproic acid and 5,7-dimethoxycoumarin induces pheomelanogenesis in the human melanoma G-361 cells, as demonstrated by Py-GC/MS/MS study

Melanogenesis is one of the main markers of the differentiation of normal and neoplastically transformed melanocytes. Valproic acid (VPA) and 5,7-dimethoxycoumarin (DMC), the potential anti-cancer drugs with differentiating activity, were reported to enhance melanin synthesis in melanoma cells. The aim of this study was to examine if the stimulation with VPA and DMC affects the structure of melanin synthesized by the human melanoma G-361 cell line. G-361 cells were cultured with VPA, DMC, and a mixture of the two agents for 7 days. Melanin isolated from the treated and untreated cells were pyrolysed, and the thermal degradation products were analyzed by gas chromatography/tandem mass spectrometry with a triple quadrupole instrument operating in a multiple reaction monitoring mode. After the stimulation with VPA and DMC, the sulfur containing pyrolytic markers of pheomelanin were detected among the thermal degradation products of the pigment isolated from the tumor cells. The levels of pheomelanin markers were the highest after the cell treatment with the combination of both agents. No sulfur compounds were present in the pyrolysate of melanin from the untreated control cells. We have concluded that VPA and DMC are able to induce pheomelanogenesis, and thus alter the structure of melanin produced in the human melanoma G-361 cells.

Abstract 3405: Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines.

Abstract Background: Resistance to targeted therapies represents a key hurdle in the treatment of cancer. Preclinical data suggest that resistance is frequently driven by redundant growth factor signaling. For example, HGF has been reported to mediate resistance to BRAF inhibitors in V600E BRAF mutant melanoma. Furthermore, clinical data support these findings as circulating and tumor HGF levels have been shown to correlate with outcome in BRAF mutant melanoma patients treated with vemurafenib. In this study, we evaluated the ability of HGF / MET signaling to attenuate the effects of BRAF and MEK inhibitors in the context of V600E BRAF and NRAS mutant cell lines. Our data suggest that combined blockade of MET and MAPK pathway signaling may be required for a robust and durable clinical response in melanoma. Methods: To confirm the ability of HGF to rescue V600E BRAF mutant melanoma cell lines from the effects of vemurafenib, G361 and SKMEL5 cells were treated with vemurafenib alone or in combination with HGF. Time lapse imaging, viability assays and cell cycle analysis were used to measure treatment effects. Analogous experiments were performed with the selective MEK inhibitor PD0325901. Combinations of a potent and selective MET inhibitor (Compound A) with vemurafenib or PD0325901 were tested to evaluate their ability to attenuate the HGF-mediated resistance. To identify other cell lines rescued by HGF, thirteen V600E BRAF mutant melanoma cell lines were treated with a dose response matrix of vemurafenib and HGF. The ability of six additional growth factors to rescue cells from the effects of vemurafenib was also tested. Three NRAS mutant melanoma cell lines were also screened for HGF-mediated resistance to PD0325901. Results: Nine of thirteen V600E BRAF mutant melanoma cell lines were rescued from the effects of vemurafenib by co-treatment with HGF. Detailed analysis of G361 and SKMEL5 cells revealed that HGF was also capable of rescuing the cells from the effects of PD0325901. This HGF-mediated rescue was attenuated by treatment with combinations of vemurafenib + compound A or PD0325901 + compound A. Mechanistic cell cycle analysis of the HGF-mediated rescue of vemurafenib in G361 cells revealed an increase in proliferating cells and a reduction in growth arrested cells, whereas rescue of PD0325901 treatment occurred through a reduction in the percentage of dead or dying cells. Of the seven growth factors tested, HGF was the predominate growth factor capable of conferring resistance to vemurafenib. Furthermore, HGF rescued one of three NRAS mutant melanoma cell lines from the effects of PD0325901, highlighting the breadth of HGF-mediated resistance in melanoma cell lines. Conclusion: These findings demonstrate the potential role for HGF / MET signaling in mediating resistance to BRAF and MEK inhibitors in melanoma, and support the clinical evaluation of MET kinase inhibitors and neutralizing antibodies to HGF in this setting. Citation Format: Sean Caenepeel, Elaina Cajulis, Rick Kendall, Angela Coxon, Paul Hughes. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2013-3405

Abstract 2509: Mechanism of anti-proliferative effects of SIRT1 inhibition in melanoma cells: a proteomics approach.

Abstract An analysis of the SEER (Surveillance, Epidemiology, and End Results) program data has suggested that the age-adjusted annual incidence of melanoma in both young men and women has increased over the past 20 years. The current therapeutic options have shown little success in melanoma management. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. The mammalian SIRT1 or sirtuin (silent mating type information regulation 2 homolog) 1 is a NAD(+)-dependent class III histone deacetylase (HDAC) that has been shown to play important roles in a variety of biological processes and diseases including cancer. Previous studies from our laboratory have demonstrated that 1) SIRT1 is overexpressed in human melanoma cells and tissues; 2) SIRT1 inhibition by Tenovin-1 resulted in decreased growth, viability, and clonogenic survival of melanoma cells; 3) Tenovin-1 treatment resulted in decreased level of SIRT1 protein and increased level of the tumor suppressor p53 (In: AACR Annual Meeting: Proceedings; 2012 March 31-April 4, Chicago, IL; Abstract # 4722). In this study we employed a proteomics approach using gel free Liquid Chromatography-Mass Spectrometry (LC-MS) to identify the downstream molecular targets of SIRT1 in melanoma. The human melanoma G361 cells were treated with Tenovin-1 (25 μM; 48 h) followed by protein extraction. Extracted proteins were precipitated (using 10% TCA and 0.05% DTT), pelleted and washed with acetone and air dried. Samples were re-suspended, labelled, trypsin digested and subjected to LC-MS/MS on a linear trap quadrupole (LTQ) Orbitrap XL. The resultant peptide sequence data were matched against the uniprot_human_FR_CP_090710 database using Mascot (Matrix Sciences, London, UK). Scaffold (version Scaffold_3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Protein identifications were accepted if they could be established at greater than 95% probability and contained at least two identified peptides. The differentially expressed protein data were collected from three different biological replicates. We found 14 proteins (from a total of 1092) which showed +2 fold differential expressions with statistical significance (T-test, p value &amp;lt;0.05). Among these proteins, three proteins were found to be newly expressed whereas one was found to completely disappear following SIRT1 inhibition, under the experimental conditions employed. The differentially expressed proteins identified in our study are associated with RNA processing-, mitochondrial checkpoint- and apoptosis- pathways. Currently, we are in process of validating and ascertaining the functional relevance of these downstream targets. Our provides an improved understanding of SIRT1 downstream targets, which may lead to better strategies for melanoma management. Citation Format: Chandra K. Singh, Jasmine George, Raj Kumar, Nihal Ahmad. Mechanism of anti-proliferative effects of SIRT1 inhibition in melanoma cells: a proteomics approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2509. doi:10.1158/1538-7445.AM2013-2509

Stat3 inhibits WTX expression through up-regulation of microRNA-370 in Wilms tumor

Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic mutations have been identified in WT patients, including inactivation of WTX, somatic stabilizing CTNNB1, and p53 mutations. However, the molecular mechanisms in tumorigenesis remain largely unexplored. Stat3 is a transcription factor that can promote oncogenesis. Stat3 activation is commonly viewed as crucial for multiple tumor proliferation and metastasis. We show that Stat3 is highly activated in Wilms tumor tissues compared to those in adjacent tissues. IL-6 treatment or transfection of a constitutively activated Stat3 in G401 cells promotes cell proliferation. At the molecular level, we further elucidate that Stat3 inhibits WTX expression through up-regulation of microRNA-370. Our results suggest that Stat3/miR-370/WTX regulatory axis might be a critical mechanism in Wilms tumor cells.

Abstract A26: Role of Dermcidin in Tumorigenesis of Skin Cancers

Abstract Dermcidin (DCD) is a candidate oncogene localized at 12q13.1. It is produced by benign melanocytic naevi and dark cells of eccrine sweat glands and proteolytic processed and transported via sweat to the epidermal surface to act as peptide antibotic, but it is not induced under inflammation. We have established the role of DCD in melanoma growth and survival by silencing DCD expression in human melanoma G361 cell line and overexpression in murine melanoma B16 cell line. Downregulation of DCD in G-361 cell line significantly reduced cellular resistance to cytotoxic agents. Balb/c Nude mice inoculated with 1x106 G-361 cells underwent a progressive increase in the tumor size and body weight loss, whereas animals inoculated with G-361-IBC I melanoma cells expressing DCD shRNA, we observed a significant difference in tumor volumes. Moreover, administration of rabbit polyclonal antibody against DCD in mice bearing G361 xenografts for four weeks delayed tumor growth. To examine tumor heterogeneity we isolated by FACS cell sorting specific cancer-stem cell populations based on cell size and enhanced tumorigenic capacity in Nude mice. In parallel, we are employing the microarray and exome methodologies to define genes that are up- and downregulated, patterns of genetic modifications and gene mutation in specific oncogenic pathways to evaluate the hypothesis that DCD control multiple oncogenic stimuli (EGFR/HER2, C-MYC, PI3K/AKT/mTOR, VEGF, BRAF/RAS, etc.). We will summarize these results and highlight genetic and biological mechanisms suggesting that DCD functions as tumor growth and cell survival factor. Citation Format: Beatriz A. Sangiuliano, Marcela Nancy Perez, Aline Cadurin, Andrew Aguiar, Dayson F. Moreira, Jose E. Belizario. Role of Dermcidin in Tumorigenesis of Skin Cancers [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A26.

머루 과피와 종자 추출물의 식품 위해성 세균에 대한 항균성 및 인체 암세포주에 대한 cytotoxicity 분석

In this study, antibacterial activity and cytotoxicity of the extracts from pericarp and seed of <TEX>$Vitis$</TEX> <TEX>$coignetiea$</TEX>, which were extracted with 0.1% HCl-60% ethanol, were analyzed. The antibacterial activity of the extracts was determined by paper disc diffusion method against food spoilage and food-borne pathogens. The pericarp extract showed high antibacterial activity against <TEX>$Bacillus$</TEX> <TEX>$cereus$</TEX>, <TEX>$Escherichia$</TEX> <TEX>$coli$</TEX> O157:H7, and <TEX>$Pseudomonas$</TEX> <TEX>$aeruginosa$</TEX>, and the seed extract represented the high antibacterial activity against <TEX>$B.$</TEX> <TEX>$cereus$</TEX>, <TEX>$E.$</TEX> <TEX>$coli$</TEX> O157:H7, and <TEX>$Staphylococcus$</TEX> <TEX>$aureus$</TEX>. The cytotoxicity of the <TEX>$Vitis$</TEX> <TEX>$coignetiea$</TEX> extract against human cancer cells was determined using the MTT assay and SRB assay. The pericarp extract represented strong growth-inhibition activity against G361 and Hep3B cells and the seed extract greatly inhibited the growth of HeLa and G361 cells in the MTT assay. In addition, the pericarp extract displayed a high inhibition activity against the growth of AGS cells and the seed extract greatly inhibited the growth of HeLa, Hep3B, and MCF7 cells in the SRB assay. Especially, the cytotoxicities of the seed extract against HeLa were significantly higher than those of the extract against other cancer cells at all test concentrations. This study demonstrates that the extract from pericarp and seed of <TEX>$Vitis$</TEX> <TEX>$coignetiea$</TEX> possess high antibacterial activity and cytotoxicity.

SOX2 contributes to melanoma cell invasion

The mechanisms of melanoma invasion are poorly understood despite extensive inquiry. SRY (sex determining region Y)-box 2 (SOX2) is an embryonic stem cell transcription factor that has recently been discovered to be expressed in human melanoma where it is associated with dermal invasion and primary tumor thickness. To assess the potential role of SOX2 expression in melanoma invasion, we examined patient melanomas and humanized melanoma xenografts, and noted preferential SOX2 expression in cells that interfaced and infiltrated dermal stroma. Experimental knockdown (KD) of SOX2 mRNA and protein in A2058 melanoma cells with high constitutive SOX2 expression resulted in 4.5-fold decreased invasiveness in vitro compared with controls (P<0.0001). Conversely, when G361 cells that normally express low SOX2 were transduced to overexpress SOX2 mRNA and protein, a 3.8-fold increase in invasiveness was observed (P=0.0004). Among 84 invasion-related genes, RT-PCR screening revealed that SOX2 KD resulted in striking decrease in matrix metalloproteinase-3 (MMP-3), an endopeptidase associated with cleavage of the extracellular matrix. Quantitatively, SOX2 KD diminished MMP-3 mRNA by 87.8%. MMP-3 KD in SOX2-expressing A2058 cells served to inhibit invasion, although to a lesser degree than SOX2 KD. Finally, immunostaining of patient and xenograft melanomas revealed coordinate SOX2 and MMP-3 expression in regions of stromal infiltration. These data implicate SOX2 expression in melanoma invasion, and suggest a role for MMP-3 as one potential mediator of this process.