C8161 Cells Research Articles

Blocking podoplanin inhibits platelet activation and decreases cancer-associated venous thrombosis

BackgroundPatients with cancer are at a high risk of venous thromboembolism (VTE), studies have shown that high expression of podoplanin (PDPN) in tumors is associated with increased risk of VTE. MethodsTwo human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN were selected to explore the role of platelet in cancer-associated venous thrombosis in vitro and in vivo. Immunohistochemical staining using anti-PDPN antibody was performed in the pulmonary carcinoma patients. ResultsBoth NCI-H226 and C8161 cells expressing high PDPN triggered platelet activation via CLEC-2 in vitro, which was abrogated by an anti-PDPN antibody SZ-168. Furthermore, the in vivo study revealed that injection of CHO-PDPN or C8161 in two mouse model of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. More importantly, PDPN-enhanced thrombosis was reduced in mice treated with SZ168. A total of 63.3% tumor specimens stained positive for PDPN. High PDPN expression was associated with an increased risk of VTE and poor prognosis. ConclusionsPDPN expression in tumors induced platelet activation and was related to a high risk of VTE via platelet activation. SZ168 inhibited PDPN-induced platelet activation in vitro and decreased the incidence of VTE in mice.

Upregulation of miR-29c-3p Hinders Melanoma Progression by Inhibiting CDCA4 Expression.

Objective To investigate the expression and regulation mechanism of miR-29c-3p and cell division cycle associated 4 (CDCA4) in melanoma (MM). Data and Methods. Fifty-nine patients with MM admitted to our hospital were enrolled as the MM group. They were followed up for 3 years to analyze the prognostic factors; meanwhile, 51 healthy subjects were allocated into a normal group. MM cell lines (M21 and C8161) were transfected with miR-29c-3p-mimics, miR-29c-3p-inhibitor, miR-NC, si-CDCA4, and sh-CDCA4. The expression of miR-29c-3p, CDCA4, Bax, Caspase3, Bcl-2, N-cadherin, vimentin, and E-cadherin was quantified, and cell proliferation, migration, invasion, and apoptosis, as well as epithelial-mesenchymal transition (EMT), were determined. Results Serum miR-29c-3p was lowly expressed and CDCA4 was highly expressed in the MM group. The area under the curve (AUC) of both for diagnosing MM was greater than 0.9. miR-29c-3p and CDCA4 were related to regional lymph node staging (N staging), distant metastasis (M staging), tumor diameter, and pathological differentiation. Low miR-29c-3p and high CDCA4 were associated with poor prognosis of MM. Overexpression of miR-29c-3p and suppression of CDCA4 hindered cell proliferation, migration, invasion, and expression of Bax, Caspase3, N-cadherin, and vimentin, but cell apoptosis and expression of Bcl-2 and E-cadherin were enhanced. Dual-luciferase reporter (DLR) assay confirmed the targeted relationship between miR-29c-3p and CDCA4. After miR-29c-3p-mimics+sh-CDCA4 was transfected into M21 and C8161 cells, the proliferation, invasion, and apoptosis were not different from those in the miR-NC group transfected with unrelated sequences. Conclusion Overexpression of miR-29c-3p suppresses CDCA4 expression and decreases proliferation, migration, invasion, apoptosis, and EMT of MM cells, thus hindering MM progression.

Role of fucosyltransferase IV in the migration and invasion of human melanoma cells.

Malignant melanoma is one of the most aggressive human tumor types, mainly due to its high invasion capability, metastatic properties, and the absence of effective treatments. Glycosylation serves a pivotal role in the migration and invasion of melanoma. However, differences in glycosylation between high and low metastatic melanoma cells and how these regulate migration and invasion by altering the expression of fucosyltransferases (FUTs) remain unclear. In the present study, matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis revealed that the composition profiling of fucosylated N-glycans differed between high metastatic C8161 and low metastatic A375P cells. Further analysis revealed that FUT4 expression was significantly increased in C8161 cells. Melanoma tissue arrays further demonstrated that FUT4 was overexpressed in metastatic samples. Altered FUT4 expression was accompanied by a change in the migration and invasion capacity of the cells. In addition, the migration and invasion potential of melanoma cells were decreased in C8161 and increased in A375P cells upon altering FUT4 expression levels by small interfering RNA or complementary DNA transfection. Furthermore, regulating FUT4 expression markedly modulated the activity of the phosphoinositide-3-kinase/Akt (PI3K/Akt) signaling pathway, which affected melanoma cell migration and invasion. In conclusion, FUT4 is a novel biomarker and regulator of the migration and invasion of melanoma cells and may serve as a therapeutic target for melanoma.

Highly Effective Drug Delivery and Cell Imaging Using Fluorescent Double-Imprinted Nanoparticles by Targeting Recognition of the Epitope of Membrane Protein.

Nanocarriers with both targeting ability and stable loading of drugs can more effectively deliver drugs to precise tumor sites for therapeutic effects. Accordingly, we have rationally designed fluorescent molecularly imprinted polymer nanoparticles (FMIPs), which use N-terminal epitope of P32 membrane protein as the primary template and doxorubicin (DOX) as the secondary template. The DOX imprinted cavity can stably carry the drug and the epitope-imprinted cavity allows FMIPs to actively recognize the P32-positive 4T1 cancer cells. The targeted therapeutic effect of DOX-loaded FMIPs (FMIPs@DOX) is investigated in vitro and in vivo. The FMIPs@DOX only causes apoptosis in 4T1 cancer cells compared to C8161 cells (expressing low level of P32). In addition, highly effective inhibition of 4T1 malignant breast tumors using FMIPs@DOX is achieved in the model of tumor-bearing mice. Importantly, the antitumor effect achieved by intravenous injection of FMIPs@DOX is almost identical to that by intratumoral injection. Furthermore, the FMIPs can serve as a targeted fluorescence imaging agent due to the high specificity of the epitope-imprinted cavity and the stable fluorescence of the embedded silicon nanoparticles. These results demonstrate the effectiveness of the FMIPs for active targeted drug delivery and imaging. Furthermore, the FMIPs provide a direction for drug-loaded nanocarrier.

Blocking podoplanin suppresses growth and pulmonary metastasis of human malignant melanoma

BackgroundPodoplanin (PDPN), a transmembrane O-glycoprotein, is up-regulated in many tumors and is involved in tumor metastasis and malignant progression. In previous studies, we generated a functional blocking monoclonal antibody (mAb, SZ168) against the extracellular domain of human PDPN. This study is aimed to investigate whether blocking PDPN by SZ168 inhibits tumor growth and metastasis.MethodsMalignant melanoma xenograft model by inoculating subcutaneously human malignant melanoma cell line C8161 into the back of BALB/c nude mice was used. Endogenous PDPN expression in C8161 cells and nasopharyngeal cancer cell line CNE-2 was detected using western blot and flow cytometry.ResultsSZ168 significantly inhibited C8161 or CNE-2 cell-induced platelet aggregation in a dose-dependent manner with a maximal inhibition of 73.9 ± 3.0% in C8161 cells or 77.1 ± 2.7% in CNE-2 cells. Moreover, SZ168 inhibited the growth and pulmonary metastasis of C8161cells in vivo. The number of lung metastatic foci in the SZ168-treated group was significantly decreased compared with that in the control mouse IgG group (1.61 ± 0.44 vs.3.83 ± 0.60, P < 0.01). Subcutaneous tumor volume, weight, and incidence were also significantly reduced in the SZ168-treated group compared to the control group (P < 0.05). Additionally, SZ168 recognized PDPN in immunohistochemical analyses of tumor tissue sections.ConclusionsSZ168 blocks growth and pulmonary metastasis of human malignant melanoma by inhibiting the interaction between tumor PDPN and platelet CLEC-2 and therefore is a promising antibody for therapeutic development against malignant melanoma.

Molecular targets for tivantinib (ARQ 197) and vasculogenic mimicry in human melanoma cells

Tivantinib (TivB) was reported previously to target MET and microtubule assembly in different cells resulting in cytotoxicity. However, its other cellular targets remain unknown, especially the proteins involved in focal adhesion and cytoskeletal organization. We studied the effect of TivB on vinculin a focal adhesion protein, and RhoC, a GTPase which promote the reorganization of cytoskeleton. Biomolecules involved in vasculogenic mimicry (VM) previously not reported in melanoma, and their susceptibility to TivB was also evaluated. TivB affects the viability and apoptosis of human melanoma cells depending on the cell type. Vinculin and RhoC were increased in the presence of TivB and affected the integrity of actin filaments and altered the cellular morphology. TivB disrupts the VM exhibited by melanoma cells in 3D matrix. Roundabout Guidance Receptor 4 (Robo4), a receptor protein implicated in axonal guidance and angiogenesis and its ligand Slit2 are expressed in human C8161 and WM793 melanoma cells, but absent in other melanoma cells including normal melanocytes. VM is more prominent in C8161 cells and could be blocked by siRNA mediated silencing of Robo4 mRNA, but TivB does not affect Robo4 in C8161 cells. Immunoblot analysis indicated no changes in Robo4 and Slit2 protein expression, however, both vinculin and RhoC protein increased in TivB treated melanoma cells. These results suggest that TivB affects cell cytoskeleton and morphology by altering proteins such as vinculin and RhoC. Our studies indicate TivB could target molecules other than MET in melanoma cells, which may provide insight into its alternate mechanism of action.

NGF reprograms metastatic melanoma to a bipotent glial-melanocyte neural crest-like precursor.

ABSTRACTMelanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type.

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

BackgroundThe metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA.ResultsIn contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine.ConclusionsThis report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Curcumin induces autophagy, inhibits proliferation and invasion by downregulating AKT/mTOR signaling pathway in human melanoma cells.

Melanoma is the foremost malignant cutaneous cancer and it is extremely resistant to chemotherapy and radiotherapy. Curcumin is an active component of turmeric, the yellow spice derived from the rhizome of Curcuma longa, and is widely known for its anti-inflammatory and anti-cancerogenic properties. Several recent studies suggest that curcumin induces apoptosis by modulating multiple signaling pathways to exert its anticancer effect. In the present study, we investigated the effect of curcumin on the viability, invasion potential, cell cycle, autophagy and the AKT, mTOR, P70S6K proteins of AKT/mTOR signaling pathway in human melanoma A375 and C8161 cell lines in vitro and in an in vivo tumorigenesis model. Curcumin effectively inhibited the proliferation of melanoma cells in vitro and in vivo. It suppressed cell invasion, arrested the cancer cells at G2/M phase of the cell cycle, and induced autophagy. Furthermore, curcumin suppressed the activation of AKT, mTOR and P70S6K proteins. Curcumin, therefore, is a potent suppressor of cell viability and invasion, and simultaneously an inducer of autophagy in A375 and C8161 cells. Accordingly, curcumin could be a novel therapeutic candidate for the management of melanoma.

Abstract 4371: Transient receptor potential cation channel 3 (TRPC3) regulates tumor proliferation and migration of BRAF wild type human malignant melanoma

Abstract Background: It has been well known that melanoma has a poor prognosis due to its rapid progression and high metastatic ability before. Although a few new drugs came out into clinical field in very recent years, these drugs are restricted to use for the patients who have BRAF or MEK gene mutation. Therefore we still have to search new target points that are not dependent on these gene mutations. TRP channels are activated by changes of temperature or membrane voltage, resulting in activation of intracellular responses. Here, we investigate whether TRPC3 regulates cell proliferation and migration of human melanoma in vitro and in vivo. Material: C8161 cells, a BRAF wild type human melanoma cell line, were mainly used in this study. Cell proliferation was assessed by 2,3, -bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) assay. Cell cycle was analyzed by fluorescence-activated cells sorting. In order to examine the role of TRPC3 in human melanoma, Short heparin RNA (shRNA) transductions with lentivirus encoding either TRPC3 or scramble was performed according to the protocols of the manufacture. Secretion level of matrix metalloproteinase (MMP) 9 was assessed in the presence of Pyr3, TRPC selective inhibitor or not by gelatin zymography. We transplanted C8161 cells into the side chest of female Balb-c nude mice at the level of intradermal depth. Immediately after tumors were formed, Pyr3 or DMSO (control) was injected intradermaly around the tumor every day. We measured each diameters of tumor every two days, and calculated volume and regression rate. Result: mRNA and protein of TRPC3 were expressed in multiple human melanoma cell lines and primary tissue. Proliferation rate of TRPC3 knocked down C8161 decreases in 48 hours (p&amp;lt;0.0001). Pyr3, one of pyrazole compounds that is known to inhibit selectively TRPC3, suppressed cell proliferation (IC50 12.99 μM). Both knockdown of TRPC3 and Pyr3 decreased path length of migration (p&amp;lt;0.01, p&amp;lt;0.01 respectively). These results suggested that TRPC3 was involved in cell proliferation and migration of C8161. Pyr3 also shortened migration path length by tracking the movement of C8161cells. Secretion level of MMP9 was decreased by Pyr3. Pyr3 inhibited phosphorylation of signal transducer and activators of transcription (STAT) 5, suggesting that TRPC3-induced proliferation and migration were regulated by, at least in part, the JAK/STAT signaling pathway. Tumor volume transplanted on mice side chest was dramatically and immediately reduced by Pyr3 local injection, while that of control group was increased. These results indicated that TRPC3 is involved in cell proliferations not only in vivo but also in vitro. Conclusion: TRPC3 regulates proliferation and migration, and thus tumor growth and metastasis of melanoma, suggesting that TRPC3 could be a novel target for treating BRAF wild type human melanomas. Note: This abstract was not presented at the meeting. Citation Format: Kayoko Oda, Masanari Umemura, Mayumi Katsumata, Haruki Aoyama, Ayako Makino, Makoto Ohtake, Itaru Sato, Yukie Yamaguchi, Yoji Nagashima, Michiko Aihara, Yoshihiro Ishikawa, Akane Nagasako. Transient receptor potential cation channel 3 (TRPC3) regulates tumor proliferation and migration of BRAF wild type human malignant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2015-4371

Effects of curcumin on the AKT/mTOR signaling pathway in human melanoma cell lines A375 and C8161

Objective To explore molecular mechanisms underlying the in vitro counteracting effect of curcumin on malignant melanoma. Methods Cultured A375 and C8161 human melanoma cells were cultivated in vitro, and randomly divided into several test groups and a control group to be treated with different concentrations of curcumin and dimethyl sulfoxide respectively for different durations. Then, methyl thiazolyl tetrazolium (MTT) assay, Transwell assay, flow cytometry and Western blot were performed to evaluate the effect of curcumin on the proliferation, invasion and cell cycle of, as well as expressions of AKT/mTOR signaling pathway-related proteins in A375 and C8161 cells respectively. Statistical analysis was carried out by using t test. Results MTT assay showed that the treatment with curcumin of 5 - 35 mg/L for 24 - 96 hours significantly inhibited the proliferation of both A375 and C8161 cells compared with that with dimethyl sulfoxide (all P< 0.001), and the inhibitory effect was in a dose-dependent manner within the range of 5 - 15 mg/L for A375 cells and within the range of 5 - 10 mg/L for C8161 cells, and in a time-dependent manner from 0 to 48 hours for both cells. After treatment for 24 hours, the 50% inhibitory concentration(IC50) of curcumin against A375 cells and C8161 cells was 10 mg/L and 5 mg/L respectively. Transwell assay demonstrated that the invasion of A375 and C8161 cells was significantly suppressed by 72-hour treatment with curcumin at 10 mg/L and 5 mg/L respectively (both P < 0.001). Flow cytometry showed that the cell cycle of A375 and C8161 cells was arrested at G2/M phase after 24-hour treatment with curcumin at 10 mg/L and 5 mg/L respectively, with significant differences in the proportion of A375 cells and C8161 cells in G2/M phase between the test group and control group (A375 cells: 35.00%±3.54% vs. 120.80%±7.46%, P < 0.001; C8161 cells: 19.33%±4.04% vs. 85.00%±9.53%, P < 0.001). Western blot revealed that the expressions of AKT/mTOR signaling pathway-related proteins were decreased in A375 and C8161 cells after 24-hour treatment with 10 mg/L and 5 mg/L curcumin respectively. Conclusion Curcumin can inhibit the proliferation and invasion of A375 and C8161 cells, likely by blocking cell cycle and inhibiting activation of the AKT/mTOR signaling pathway. Key words: Melanoma, experimental; Curcumin; Cell proliferation; Cell cycle; Signal transduction

Inhibitory effect of apigenin on proliferation and invasion of human malignant melanoma cells

Objective To evaluate the inhibitory effect of apigenin on malignant melanoma in vitro, and to investigate its mechanisms. Methods Human malignant melanoma cell lines A375 and C8161 were divided into test groups and control group separately. Cells in the test groups were treated with apigenin at different concentrations, while cells in the control group were treated with dimethyl sulfoxide, for different durations. Subsequently, methyl thiazolyl tetrazolium（MTT）assay, wound healing assay and Matrigel invasion assay were carried out to estimate cellular proliferative activity, migratory activity and invasive activity, respectively, and scanning electron microscopy（SEM）was used to observe morphology of melanocyte dendrites. Flow cytometry using annexin-V/propidium iodide（PI）staining was performed to detect cell apoptosis, propidium iodide（PI）staining to analyze cell cycle, and Western blot to measure the expressions of proteins related to apoptosis and involved in the extracellular signal-regulated kinase（ERK）signaling pathway. Results MTT assay showed significant differences in cellular proliferative activity between the test groups and control group（all P < 0.05）. The proliferation of A375 and C8161 cells was inhibited by apigenin in a dose-dependent manner when the concentration of apigenin was 10-40 mg/L, and in a time-dependent manner when the treatment duration varied from 0 to 48 hours. The half-maximal inhibitory concentration（IC50）of apigenin at 24 hours was 25 mg/L for both A375 and C8161 cells. Wound healing assay showed that the migration of A375 and C8161 cells was significantly decelerated after 24-hour treatment with apigenin of 10, 20 and 25 mg/L compared with the control cells（all P < 0.01）. Matrigel invasion assay demonstrated that the number of invasive cells was significantly smaller in A375 and C8161 cells treated with apigenin of 10, 20 and 25 mg/L for 72 hours than in the control cells（all P< 0.01）. SEM showed that the dendrits of both A375 and C8161 cells became thinner and longer after treatment with 25 mg/L apigenin for 24 hours, with the length of dendrits being（23.30 ± 2.62）μm and（16.50 ± 1.62）μm respectively, compared to（12.38 ± 2.27）μm and（9.36 ± 2.51）μm respectively in the control groups（both P < 0.01）. After treatment with apigenin of 10 and 25 mg/L for 24 hours, a significant increase was observed in apoptosis rate in both A375 cells（3.30% ± 0.82% vs. 0.40% ± 0.07%, P< 0.01; 10.00% ± 0.60% vs. 4.00% ± 0.70%, P< 0.01）and C8161 cells（13.10% ± 1.45% vs. 7.27% ± 1.31%; 25.77% ± 2.40% vs. 7.27% ± 1.31%; both P < 0.01）compared with the control cells. Both A375 and C8161 cells were arrested in G2/M phase after treatment with 25 mg/L apigenin for 24 hours, with the percentage of cells in G2/M phase being 48.70% ± 3.04% and 31.10% ± 1.90% respectively, compared to 21.30% ± 0.75% and 25.06% ± 2.12% respectively in the control groups（both P< 0.01）. Western blot showed an increase in the expressions of apoptosis-related proteins including cleaved caspase-3 and cleaved poly ADP-ribose polymerase（PARP）with the activation of ERK signaling pathway in both A375 and C8161 cells after 24-hour treatment with 25 mg/L apigenin compared with the control groups. Conclusions Apigenin can inhibit the proliferation, migration and invasion of, but induce apoptosis and cell cycle arrest in human malignant melanoma cells, likely by regulating the expression of ERK signaling pathway-related proteins. Key words: Apigenin; Melanoma, experimental; Cell proliferation; Cell movement; Extracellular signal-regulated MAP kinases

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (⩽1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (⩽1% oxygen) and hypoxia mimetic CoCl2. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5mM glucose after 48h of physical hypoxia. However, combining the latter with 100μM CoCl2 was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl2 in 5mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.

Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

BackgroundThe Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines.MethodsThe A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms.ResultsODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells.ConclusionsThe apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior.

Abstract 5244: Store-operated Ca2+ entry (SOCE) regulates melanoma progression.

Abstract Background: Melanoma is the worst poor-prognosis skin cancer in the U.S. Although Ca2+ is well recognized as a major second messenger, the role of Ca2+ remains poorly understood in the cancer research field including melanoma. Store-operated calcium entry (SOCE) is the mechanism of Ca2+ entry from the extracellular space in response to Ca2+ depletion in the endoplasmic reticulum (ER). SOCE is regulated by interaction between STIM1 (stromal interaction molecule 1) in the ER and Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane. Our previous studies have demonstrated the presence of SOCE is observed in melanoma cells, and potentially regulates cell migration and cell cycle. In the present study, we extensively examined the effects of SOCE on migration and proliferation in multiple melanoma and melanocyte cell lines as well as on metastasis in mice. Method: Changes in intracellular Ca2+ level were measured by Fluo4-AM, a Ca2+-sensing fluorescent dye. In order to ablate STIM-1 or Orai-1, shRNA for each protein was induced with lentiviral infection in SK-Mel-2, SK-Mel-24 and C8161 cells. Proliferation was measured by the MTT assay. Migration was examined with the Boyden chambers and the scratch assay. Immunocytochemistry was performed to count the number of lamellipodia with F-actin staining. The experimental metastasis assay was performed as follows. Three weeks after the intravenous injection of SK-Mel-2 cells with knockdown of Orai1 or STIM1 in Balb/c nu/nu mice, the lungs were removed and fixed with picric acid. The number of metastatic colonies in the lung surface was counted. Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in 5 primary and 3 metastatic human melanoma cell lines, a mouse melanoma cell line, human and mouse melanocyte cell lines. In addition, metastatic, but not primary, melanoma cell lines showed significantly greater SOCE compared to melanocytes, suggesting that SOCE may positively correlate with malignancy of melanoma. Knockdown of STIM1 inhibited proliferation in metastatic melanoma cells (SK-Mel-2, SK-Mel-24 and C8161) (p&amp;lt;0.01), suggesting that SOCE activates proliferation of melanoma. Inhibition of SOCE with knockdown of Orai1 or STIM1 suppressed migration in cell lines tested (SK-Mel-2 and SK-Mel-24) (p&amp;lt;0.01) and the SOCE inhibitors suppressed it in cell lines (SK-Mel-2 and C8161) (p&amp;lt;0.01 for both cell lines), indicating that SOCE regulates melanoma migration. Indeed, the number of lamellipodia, which reflects the activity of migration, was decreased by deletion of STIM1 or Orai1 (p&amp;lt;0.01). The experimental metastasis assay demonstrated that the number of lung colonies was reduced by knockdown of STIM1 or Orai1 (p&amp;lt;0.01). Conclusion: Our results demonstrated that inhibition of SOCE suppressed proliferation, migration, and metastasis in melanoma, thus SOCE could be a new target for melanoma therapy. Citation Format: Masanari Umemura, Erdene Balijinnyam, Stefan Feske, Mariana S. De Lorenzo, Lai-Hua Xie, Yoshihiro Ishikawa, Kousaku Iwatsubo. Store-operated Ca2+ entry (SOCE) regulates melanoma progression. [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 5244. doi:10.1158/1538-7445.AM2013-5244

Lycorine hydrochloride inhibits metastatic melanoma cell‐dominant vasculogenic mimicry

Melanoma cells actively participate in tumor angiogenesis and vasculogenic mimicry. However, anti-angiogenic therapy in patients with melanoma has not shown a significant survival gain. Thus, new anti-melanoma angiogenic and vasculogenic drugs are highly desired. Using the metastatic melanoma cell line C8161 as a model, we explored melanoma vasculogenic inhibitors and found that lycorine hydrochloride (LH) effectively suppressed C8161 cell-dominant formation of capillary-like tubes in vitro and generation of tumor blood vessels in vivo with low toxicity. Mechanistic studies revealed that LH markedly hindered expression of VE-cadherin in C8161 cells, but did not affect expression of six other important angiogenic and vasculogenic genes. Luciferase assays showed that LH significantly impeded promoter activity of the VE-cadherin gene in a dose-dependent manner. Together, these data suggest that LH inhibits melanoma C8161 cell-dominant vasculogenic mimicry by reducing VE-cadherin gene expression and diminishing cell surface exposure of the protein.

Abstract 1864: Role of store-operated Ca2+ entry in proliferation and cell cycle in melanoma

Abstract Background: Melanoma has a poor prognosis due to its rapid progression and a high metastatic ability. Ca2+ homeostasis plays a pivotal role in proliferation in cancer cells, but little is known about the role of Ca2+ in melanoma. Store-operated calcium entry (SOCE) is defined as Ca2+ entry from extracellular space triggered when Ca2+ store in the endoplasmic reticulum (ER) is depleted. SOCE is regulated by coordination between Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane, and STIM1 (stromal interaction molecule 1) in the ER. We examined the role of Ca2+ in melanoma cells especially focusing on SOCE. Method: Intracellular Ca2+ level was measured by using Fluo4-AM, a Ca2+-sensing fluorescent dye. Cell proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were stained with propidium iodide and cell cycle was assessed using flow cytometer. Protein phosphorylation microarray was performed by using a commercially available kit (Cancer Signaling Phospho-Antibody Array). Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in SK-MEL-2, C8161, SK-MEL-24, UACC257 (human) and B16 (mouse) melanoma cell lines, suggesting that the existence of SOCE is universal among melanoma cells. SOCE was attenuated by Pyr3, a pyrazole compound which is known to inhibit SOCE, as well as by EGTA that chelates extracellular Ca2+. Pyr3 inhibits cellular proliferation in SK-MEL-2 cells (IC50: 5.1uM) and C8161 cells (IC50: 2.1uM). Cell cycle was analyzed in C8161 cells, and the cell number in the G2 phase was increased by both Pyr3 and EGTA, indicating that SOCE plays a role in the transition of G2/S phase. Protein microarray data showed that Pyr3 reduces phosphorylation of signal transducers and activators of transcription 5 (STAT5) by 93%, suggesting that the JAK/STAT pathway may be involved in SOCE-induced alteration of cell cycle. Conclusion: Our results showed that inhibition of SOCE suppresses proliferation in melanoma, thus it could be a new strategy for melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1864. doi:1538-7445.AM2012-1864

Abstract 4302: Odontogenic ameloblast-associated protein (ODAM) alters the growth and migration of human melanoma cells

Abstract Initial studies of the Odontogenic Ameloblast-Associated Protein (ODAM) revealed that it is highly expressed in mature ameloblasts and is present in the rodent enamel organ and junctional epithelium. Further analysis showed that ODAM is expressed in several epithelial malignancies including gastric, colon, breast, lung and melanoma. A correlation was observed between ODAM expression/localization and breast cancer stage/clinical patient outcome, indicating that ODAM may serve as a novel prognostic biomarker in this type of cancer. When transfected with rODAM and injected into mice intravenously, the MDA-MB-231 breast cancer cell line showed marked inhibition of neoplastic and metastatic properties. This suggests that ODAM has a potentially significant regulatory role in tumorigenesis and metastasis of breast cancer with important clinical implications. To investigate the role of ODAM in melanoma, archived slides prepared from primary melanoma tissue specimens were obtained and stained with anti-ODAM antibodies and results were compared with retrospective clinical data. In patients that were found to have positive sentinel lymph nodes, ODAM localized to the nucleus in 76% of the cases. This suggests that nuclear-localization of ODAM in the primary tumor is a negative prognostic factor, which could assist in determining management of this devastating cancer. Melanoma is frequently diagnosed at late stages, often metastatic to regional lymph nodes or distant sites. The inhibition of neoplastic and metastatic properties shown in ODAM-expressing breast malignancies encouraged further investigation into the effect of ODAM expression in the tumorigenesis of melanoma. The human metastatic melanoma cell line C8161 does not express ODAM and was stably transfected with rODAM. In two consecutive growth assays, the ODAM-expressing C8161-clones showed significant growth inhibition compared to parent C8161 cells. In wound-healing assays of cell migration, exogenous rODAM was added at increasingly higher concentrations to wells of C8161 cells at 100% confluency immediately before scratch-wounding the monolayer. After twenty-four hours, C8161 parent cells had near-complete coverage of the scratch defect. In the wells with added exogenous rODAM, cells showed a decreased ability to migrate and cover the scratch, with more inhibition of migration as the concentration of rODAM increased from 3 to 12 µg/mL. These findings demonstrate that ODAM alters the behavior of the C8161 human melanoma cell line with respect to growth and cell migration. Further studies to assess tumorigenesis in vivo are under way, concerning the potential effect that ODAM may have on invasion, gene expression and metastatic characteristics of this cell line. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4302. doi:1538-7445.AM2012-4302

Mda-9/Syntenin Protein Positively Regulates the Activation of Akt Protein by Facilitating Integrin-linked Kinase Adaptor Function during Adhesion to Type I Collagen

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.

Epac1 promotes melanoma metastasis via modification of heparan sulfate

Our previous report suggested the potential role of the exchange protein directly activated by cyclic AMP (Epac) in melanoma metastasis via heparan sulfate (HS)-mediated cell migration. In order to obtain conclusive evidence that Epac1 plays a critical role in modification of HS and melanoma metastasis, we extensively investigated expression and function of Epac1 in human melanoma samples and cell lines. We have found that, in human melanoma tissue microarray, protein expression of Epac1 was higher in metastatic melanoma than in primary melanoma. In addition, expression of Epac1 positively correlated with that of N-sulfated HS, and N-deacetylase/N-sulfotransferase-1 (NDST-1), an enzyme that increases N-sulfation of HS. Further, an Epac agonist increased, but ablation of Epac1 decreased, expressions of NDST-1, N-sulfated HS, and cell migration in various melanoma cell lines. Finally, C8161 cells with stable knockdown of Epac1 showed a decrease in cell migration, and metastasis in mice. These data suggest that Epac1 plays a critical role in melanoma metastasis presumably because of modification of HS.