Human Oral Carcinoma Cell Lines Research Articles

Pharmacokinetic characteristics and anticancer effects of 5-Fluorouracil loaded nanoparticles

BackgroundIt is expected that prolonged circulation of anticancer drugs will increase their anticancer activity while decreasing their toxic side effects. The purpose of this study was to prepare 5-fluorouracil (5-FU) loaded block copolymers, with poly(γ-benzyl-L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene glycol) (PEG) as the hydrophilic block, and then examine the 5-FU release characteristics, pharmacokinetics, and anticancer effects of this novel compound.Methods5-FU loaded PEG-PBLG (5-FU/PEG-PBLG) nanoparticles were prepared by dialysis and then scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the shape and size of the nanoparticles, and ultraviolet spectrophotometry was used to evaluate the 5-FU in vitro release characteristics. The pharmacokinetic parameters of 5-FU/PEG-PBLG nanoparticles in rabbit plasma were determined by measuring the 5-FUby high-performance liquid chromatography (HPLC). To study in vivo effects, LoVo cells (human colon cancer cell line) or Tca8113 cells (human oral squamous cell carcinoma cell line) were implanted in BALB/c nude mice that were subsequently treated with 5-FU or 5-FU/PEG-PBLG nanospheres.Results5-FU/PEG-PBLG nanoparticles had a core-shell spherical structure with a diameter of 200 nm and a shell thickness of 30 nm. The drug loading capacity was 27.1% and the drug encapsulation was 61.5%. Compared with 5-FU, 5-FU/PEG-PBLG nanoparticles had a longer elimination half-life (t1/2, 33.3 h vs. 5 min), lower peak concentration (C, 4563.5 μg/L vs. 17047.3 μg/L), and greater distribution volume (VD, 0.114 L vs. 0.069 L). Compared with a blank control, LoVo cell xenografts and Tca8113 cell xenografts treated with 5-FU or 5-FU/PEG-PBLG nanoparticles grew slower and had prolonged tumor doubling times. 5-FU/PEG-PBLG nanoparticles showed greater inhibition of tumor growth than 5-FU (p < 0.01). In the PEG-PBLG nanoparticle control group, there was no tumor inhibition (p > 0.05).ConclusionIn our model system, 5-FU/PEG-PBLG nanoparticles changed the pharmacokinetic behavior of 5-FU, thus increasing its anticancer activity. 5-Fluorouracil loaded nanoparticles have potential as a novel anticancer drug that may have useful clinical applications.

Retinoic Acid and Sodium Butyrate as Cell Cycle Regulators in the Treatment of Oral Squamous Carcinoma Cells

All-trans retinoic acid (ATRA) and sodium butyrate (SB) have shown growth-inhibitory and differentiation-inducing properties to tumor cells when used as single agents or in combination, but the exact molecular mechanism still remains to be determined. In order to determine the mechanism of the synergy in treatment with RA and SB, we evaluated the growth inhibition capability of ATRA and SB, alone or in combination, in human oral squamous carcinoma cell lines SCC-1 and SCC-9, and identified the expression of cell cycle-related genes. ATRA and SB inhibited cell growth and induced cell cycle G1 arrest. The inhibition effect was more pronounced with SB than with ATRA (p = 0.000). There were interactions between ATRA and SB (p = 0.000). Consistent with the inhibition effect and G1 arrest, ATRA and SB, alone or in combination, induced the expression of G1 phase markers cyclin-dependent kinase (CDK) 6, p21, and p27; inhibited the expression of S-G2 phase proteins CDK2; and decreased Rb phosphorylation. Cyclin D1 expression was increased in the SB- and ATRA + SB-treated groups, but inhibited in the ATRA-treated group. Cyclin B1 and cyclin E expression was slightly decreased in the SB- and ATRA + SB-treated groups, but did not change in the ATRA-treated group. These results indicate that the growth inhibition and G1 arrest of oral squamous carcinoma cells in response to ATRA and/or SB correlates with the induction of G1 phase cell cycle regulatory proteins CDK6, p21, and p27 and the inhibition of S-G2 phase cell cycle regulatory protein CDK2.

Two new sesquiterpenoids from the soft coral Sinularia polydactyla (Ehreberg)

Two new sesquiterpenoids, polydactins A (1) and B (2) and a known sesquiterpene, 10α-hydroxycadin-4-en-15-al (3), were isolated from the soft coral Sinularia polydactyla (Ehreberg). Their structures were determined mainly by spectroscopic methods. Polydactin A (1) showed moderate cytotoxic activities against human oral epidermoid carcinoma cell lines (KB) and human breast carcinoma (MCF) tumour cell lines (in vitro).

Anti‐metastasis effect of PA‐22 in oral squamous cell carcinoma HSC‐3 cells

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. In the present study, human oral squamous cell carcinoma (OSCC) cell lines HSC‐4 were used to evaluate the effects of PA‐22 on the cell cycle and the invasion activity. PA‐22, is isolated from Chinese medicine herb physalis anagata with respect to tumor growth, invasion in OSCC model. PA‐22 treated HSC‐4 cells dose and time‐dependently by MTT assay, Flow cytometry, DAPI assay, DNA Ladder assay, Comet assay, and Western blot. PA‐22 treatment triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl‐2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and active caspases. We also found Matrigel invasion assay revealed that the invasiveness of HSC‐3 was suppressed by treatment with PA‐22. It down‐regulate the secretion of matrix metalloproteinase (MMP) to culture supernatant as well as the expression of MMP proteins. These results indicate that PA‐22 is a novel candidate agent for the treatment of oral cancer.

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in YD9 Human Oral Squamous Carcinoma Cells

Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21 WAF1/CIP1 and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.

The effect of hypoxic microenvironment on matrix metalloproteinase expression in xenografts of human oral squamous cell carcinoma

Hypoxia is a common environmental stress factor and is associated with physiological and pathological conditions related to cancer invasion and metastasis. The process of cancer cell invasion involves degradation of the extracellular matrix. Here, we examine the effect of hypoxic microenvironment on matrix metalloproteinase expression in human oral squamous cell carcinoma under in vitro and in vivo conditions. At first, the expression levels of HIF-1alpha and matrix metalloproteinase (MMP) proteins in human oral squamous cell carcinoma cell lines, SAS and HSC-2 cultured under hypoxic or normoxic condition, were assessed by Western blotting. Enzyme activity and mRNA of MMP under hypoxic or normoxic condition were also investigated. Then the SAS and HSC-2 cells were transplanted subcutaneously into immunodeficient mice and the correlation between hypoxia and protein expression for MMPs, HIF-1alpha and Ki-67 were assessed. Hypoxic region was detected by in situ hypoxic probe, pimonidazole. MMP proteins and mRNA in both SAS and HSC-2 cells were increased under hypoxic condition. In xenograft, MMP-2 was expressed in tumor tissue, especially in hypoxic region. In contrast, MMP-9 expression was recognized in tumor tissue, especially neighboring stromal tissues containing blood vessels. Our study suggests that the hypoxic microenvironment in human oral squamous cell carcinoma plays important roles in expression for HIF-1alpha and MMPs, and proliferative activity of tumor cells.

Nitrogen-Containing Verticillene Diterpenoids from the Taiwanese Soft Coral Cespitularia taeniata

Extensive column chromatography of the ethanolic extract of the soft coral Cespitularia taeniata collected in Taiwan has resulted in the isolation of eight new nitrogen-containing verticillene diterpenes, designated cespitulactams D-K ( 1- 8), together with three known diterpenes, cespitulactams A ( 9) and B ( 10) and cespitularin F. In addition, one new derivative, 6- O-acetylcespitulactam F ( 11), was prepared from compound 3. The structures of these compounds were elucidated on the basis of spectroscopic analyses, especially HRMS and 2D NMR experiments. The cytotoxicity against human oral epidermoid carcinoma (KB) and murine L1210 leukemia cell lines and antimicrobial activities of 1- 8 and 11 were tested and evaluated. A biogenetic pathway for these novel diterpene alkaloids was also proposed.

Epigenetic regulation of chemosensitivity to 5-fluorouracil and cisplatin by zebularine in oral squamous cell carcinoma

Epigenetic alterations such as histone acetylation and DNA methylation play an important role in the regulation of gene expression for cell cycles and apoptosis that may affect the chemosensitivity of cancers. Previously, we have reported that the combination of suberoylanilide hydroxamic acid (SAHA), a newly developed histone deacetylase inhibitor, with cisplatin (CDDP) possessed synergistic cytotoxicity against human oral squamous cell carcinoma (OSCC) cell line HSC-3. In this study, we used a novel DNA methyltransferase inhibitor, zebularine (Zeb), to investigate the epigenetic influence on the sensitivity of carcinoma cell lines to 5-fluorouracil (5-FU) or CDDP by evaluating apoptotic inducibility. Treatment with CDDP or 5-FU either alone or in combination with Zeb or SAHA continued for 48 or 72 h. In HSC-3 cells, Zeb had chemosensitive efficacy with CDDP, but not 5-FU, whereas SAHA showed efficacy with both CDDP and 5-FU. We showed that Zeb has strong anti-proliferative activity against HSC-3 cells, shown by decreased cellular growth and G2/M cell cycle phase accumulation. Furthermore, DNA methylation could be a regulatory mechanism for dihydropyrimidine dehydrogenase (DPD), known to be a principal factor in 5-FU resistance. CDHP (5-chloro-2,4-dihydroxypyridine), an inhibitor of DPD, had an enhancing effect on the apoptotic ability of 5-FU alone or 5-FU/Zeb combination. In conclusion, the present study suggests that low-dose (IC20) Zeb may sensitize cancer cells to CDDP, which may be an important characteristic for solid cancer treatment, and that DPD and other agents activated by Zeb in cancer cells could be an inhibitory factor in the response to apoptosis induced by 5-FU.

Synthesis, Crystal Structure, Cytotoxic and Apoptotic Activity of 2,4-Dichloro-6-methylquinoline on Human Oral Carcinoma Cell Line

The present report describes the synthesis, IR spectra, 3-dimensional structure of the compound 2,4-dichloro-6-methylquinoline and evaluation of its anti-cancer activity using propidium iodide (PI) staining and annexin binding assay techniques. This derivative of quinoline was synthesized from the mixture of p-toluidine and malonic acid and synthesis has been achieved in a one-pot reaction from an aryl amine, malonic acid and phosphorous oxychloride. Crystallographic data reveals that the crystals belong to triclinic crystal system with space group P-1 with the unit cell dimensions of a = 7.14(1) A, b = 11.53(1) A, c = 11.97(1) A and α = 90.18° (10), β = 106.31° (10), γ = 91.07° (10). The in vitro anti-cancer assay indicated that compound has cytotoxic and apoptotic activity on human oral squamous carcinoma (KB) cell line, thus it could be developed as a potent anti-cancer agent. Synthesis, Crystal Structure, Cytotoxic and Apoptotic Activity of 2,4-Dichloro-6-methylquinoline on Human Oral Carcinoma Cell Line R. K. Somvanshia, R. Subashinib, V. Dhanasekaranb, G. Arulprakashb, S. N. Dasc, S. Deya

Chemokine Receptor Expression in Oral Squamous Cell Carcinoma: Correlation with Growth Factor- and Cytokine-mediated Cell Migration in vitro

Metastasis is the chief cause of mortality in cancer patients. Recently, chemokines and chemokine receptors were shown to play an important role in the metastasis of various cancers. We examined the role of chemokine receptor-mediated signaling in the invasion potential of human oral squamous cell carcinoma (OSCC) cell lines that were derived from 5 primary tumors and 6 cervical lymph node metastases. Comprehensive analysis of the mRNAs for human chemokine receptors showed that the OSCC cell lines had uniform expression patterns of chemokine receptors. Overall, there were no consistent differences in the expression of chemokine receptors between primary site- and lymph node metastasis-derived cell lines. However, a highly invasive OSCC cell line (SAS-H1) expressed up-regulation of CCR5, CCR6, CCR7, CXCR1, CXCR6 and CX3CR1 compared to a poorly invasive OSCC cell line (SAS-L1). Then we examined whether factors in the tumor microenvironment regulated chemokine receptor expression in SAS-H1 cells. Specifically, transforming growth factor (TGF) -β1 enhanced the expression of CCR5, CCR6, CCR7 and CX3CR1. Pretreatment of SAS-H1 cells with transforming growth factor (TGF) -β1 increased the expression of CCR7 and CX3CR1, and then enhanced CCL21- and CX3CL1-induced directional migration (1.5-fold enhancement as compared with untreated control). In addition, CX3CL1 increased the adhesion of SAS-H1 cells on uncoated tissue culture plates. Neither chemokine stimulated cell proliferation. Treatment of SAS-H1 cells with CX3CL1 activated the phosphotidylinositol-3-kinase (PI3K) and MEK signal transduction pathways. Our results suggest that chemokine receptor-mediated signaling is involved in the local invasion and metastasis of human OSCC.

Upregulation of IGF-2 and IGF-1 receptor expression in oral cancer cell lines

The insulin-like growth factors (IGFs) are a family of mitogenic proteins involved in the regulation of cell growth and differentiation. The presence and role of the IGF system in oral mucosal epithelium is not clear but could influence our understanding of the pathogenesis of oral cancer. We characterised the expression and function of IGF-1, IGF-2 and IGF receptor in human oral squamous carcinoma cell lines and normal oral epithelial cells as well as normal oral and squamous cell carcinoma tissues. Using reverse transcription followed by PCR, IGF-1 mRNA was only detected in normal cells, whereas IGF-2 and IGF-1R mRNA transcripts were highly expressed in tumour cell lines and tissues. Similar observations were seen by Western blot analysis and immunohistochemistry. Exogenous IGF-2, but not IGF-1, caused significant increases in DNA synthesis in the cell lines. IGF-2 also increased cell proliferation which was significantly attenuated in the presence of an IGF-2 neutralizing antibody or one which blocked IGF-1R. Taken together, these studies suggest that autocrine production of IGF-2, together with over-expression of IGF-1R, may be important components controlling the proliferation of oral carcinoma cells.

Expression of p21 and p53 in rat gingival and human oral epithelial cells after cyclosporine A treatment

Expression of p21 and p53 were examined, at gene and protein levels, in edentulous gingival epithelial cells from rats and from a human oral epidermoid carcinoma cell line, OECM1, after cyclosporine A therapy. In vivo: 20 partially edentulous SD rats were assigned into cyclosporine A feeding and control groups. After the rats were killed, p21 and p53 in gingiva were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. In vitro: after cyclosporine A treatment, p21 and p53 of OECM1 cells were evaluated by western blot and the luciferase assay. The distribution of OECM1 cells in each phase of the cell cycle was evaluated by flow cytometry. The mRNA expression of p21 was significantly higher in the cyclosporine A group than in the control group. A greater number of positive anti-p21-stained cells were observed in the gingival epithelium of the cyclosporine A group than in the control group. Significantly higher levels of p21 protein and activity were observed in OECM1 cells after cyclosporine A treatment than in cells without treatment. A relative increase of cells in G0/G1 phases, and a decrease of cells in G2/M phases, were observed in OECM1 cells after cyclosporine A treatment. In the present study, higher p21 mRNA and protein expressions were observed after cyclosporine A treatment. Thus, an up-regulation of p21 expression, via a p53-independent pathway, by cyclosporine A in gingival and oral epithelial cells was suggested.

Poster 076: Consideration of the Expression of VEGF by Photodynamic Therapy and Examination to Clinical Application

Photodynamic therapy (PDT) has been demonstrated as a useful method for treating precancerous and cancer lesions of skin, bladder and oral cavity. However, recurrence of the tumor after PDT remains problematic in spite of a good initial response. In this regard, some studies have shown that PDT induces vascular endothelial growth factors (VEGF) expression in human oral/other organs squamous cell carcinoma. However, little is known about VEGF expression applied PDT in human carcinoma cell line. There have been no reports about PDT using Npe6 (Npe6-mediated PDT), a second generation photosensitizer, in human oral carcinoma cell line, HSC-3 cells. We investigated the expression of VEGF, c-jun and c-fos protooncogenes in HSC-3 cells in response to Npe6-mediated PDT. We also addressed the possibility of oxidative damage induced by PDT could lead to angiogenic response, via VEGF expression, and we investigated the expression of VEGF in tongue carcinoma, gingival carcinoma, and thyroid carcinoma in response to COX-2 inhibitor. n/a RT-PCR analysis reveled the Npe6-mediated PDT induced the expression of mRNAs for VEGF, c-jun and c-fos in time- and concentration-dependent manners. Desferrioxamine (DFX), an iron chelator, induced the VEGF expression, but the expression pattern was different from that of Npe6-mediated PDT. The expression mRNAs for VEGF, c-jun and c-fos induced by Npe6-mediated PDT was inhibited by SB203580, p38 MAPK inhibitors, and the expression of VEGF mRNA was inhibited by cycloheximid (CHX), a protein synthesis inhibitor. The c-jun mRNA expression was inhibited whereas the c-fos mRNA expression was enhanced by N-acetyl-Lcysteine (NAC), a free radical scavenger. We conclude that Npe6-mediated PDT induces the expression of VEGF, c-jun and c-fos in human oral carcinoma cell line, HSC-3 cell, at least partly, through the activation of p38 MAPK. Suppression of VEGF expression was expected to clinical application. Now, we further examine the effectiveness of COX-2 inhibitor.

Poster 057: Metastasis-Associated MTA1 Expression in Oral Squamous Cell Carcinoma: Relationship With Lymph Node Metastasis

Metastasis-associated protein 1 (MTA1) is physiologically expressed at only lowlevels in human tissue, except testis. Its expression has been found to be associated with progression in solid cancers of various organs and cancer cell lines with high invasive potential and thus is believed to play a role in cancer progression to the metastatic state. Functional studies supported this by showing that experimental overexpression of MTA1 in keratinocytes is associated with increase in migration and invasion and survival in the anchorage-independent state. However, few reports have examined the role of MTA1 in oral squamous cell carcinoma. To determine the relationship between clinicopathologic factors and MTA1 expression in patients with oral squamous cell carcinoma, we investigated MTA1 gene expression and protein production. 38 oral squamous cell carcinomas (tongue carcinoma) were enrolled in this study. The expression of MTA1 was assayed immunohistochemically using polyclonal antibodies, and the expression of PCNA was assayed immunohistochemically using monoclonal antibody. The human oral squamous cell carcinoma cell lines, SAS, HSC-2, OSC19 and OSC20 were obtained from the Human Science Research Resource Bank. For PCR analysis, cDNA was amplified by Taq DNA polymerase. BioCoat Matrigel invasion chambers were used to assess cell invasion. Briefly, cells were seeded in the upper chamber. Cells on the reverse side of the insert were fixed and stained with DiffQuick. To investigate the correlation of MTA1 with cancer invasion, the effect of MTA1 inhibition was examined by RNA interference (RNAi). Significance was assessed by means of Student T test and Pearsons correlation coefficient. MTA1 protein expression was detected in 33 of 38 SCCs. There was no correlation between MTA1 expression and T category. There was correlation between MTA1 expression and N category and clinical stage. Immunohistochemical expression of MTA1 had no correlation with tumor cell growth factor (PCNA) and WHO histological malignancy grade. MTA1 protein production was strongly associated with cancer cell invasion (p<0.05), and there was significant correlation with lymph node metastasis and MTA1 protein production. HSC2 revealed low mRNA expression and OSC 20 revealed highest mRNA expression. In matrigel invasion assay, the HSC2 cell line revealed lowest invasion and the OSC20 cell line revealed highest invasion. RNAi-mediated MTA1 reduction decreased the invasion index (p<0.01). MTA1 may be a useful marker for lymph node metastasis, and MTA1 expression may be associated with increased invasive ability in oral squamous cell carcinomas.

Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Comparative Analysis of Gene Expression Profiles of 3 Different Human Oral Squamous Carcinoma Cell Lines With Different Invasion and Metastatic Capacities

Squamous cell carcinoma is the most common cancer of the oral cavity. There is little published information about how oral squamous cell carcinoma (OSCC) invades the jaw bones and metastasizes to cervical lymph nodes and/or distant organs. In this study, 3 OSCC human cell lines, BHY, HN and HSC-3, were chosen to study based on their known biologic profiles. The working hypothesis of this study was that OSCCs with different behaviors have different gene expression profiles.

Poster 039: Isolation and Characterization of a Side Population of Human Oral Squamous Cell Carcinoma Cell Line, Ho-1-N-1

Poster 039: Isolation and Characterization of a Side Population of Human Oral Squamous Cell Carcinoma Cell Line, Ho-1-N-1

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines

Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 μg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630 ± 20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.

Alvaradoins E-N, antitumor and cytotoxic anthracenone C-glycosides from the leaves of Alvaradoa haitiensis.

Bioactivity-directed fractionation of an extract of the leaves of Alvaradoa haitiensis, using the KB (human oral epidermoid carcinoma) cell line, led to the isolation and identification of 10 new anthracenone C-glycosides, alvaradoins E-N (1-10), along with the known compound chrysophanol (11). The cytotoxicity of all compounds was evaluated, and preliminary structure-activity relationships are suggested. The most potent compounds in the in vitro assays (1 and 2) were evaluated in vivo versus the P388 (murine lymphocytic leukemia) model, and alvaradoin E (1) showed antileukemic activity (125% T/C) at a dose of 0.2 mg kg-1 per injection when administered intraperitoneally.

Galanin and galanin receptor type 1 suppress proliferation in squamous carcinoma cells: activation of the extracellular signal regulated kinase pathway and induction of cyclin-dependent kinase inhibitors

Galanin receptor 1 (GALR1) maps to a common region of 18q loss in head and neck squamous cell carcinomas and is frequently inactivated by methylation. To investigate effects of GALR1 and its signaling pathways, we stably expressed hemaglutinin-tagged GALR1 in a human oral carcinoma cell line (UM-SCC-1-GALR1) that expresses no endogenous GALR1. In transfected cells, galanin induced activation of the extracellular-regulated protein kinase-1/2 (ERK1/2) and suppressed proliferation. Galanin stimulation mediated decreased expression of cyclin D1 and increased expression of the cyclin-dependent kinase inhibitors (CKI), p27(Kip1) and p57(Kip2). Pretreatment with the ERK1/2-specific inhibitor U0126 prevented these galanin-induced effects. Phosphatidylinositol 3-kinase (PI3K) pathway activation did not differ in UM-SCC-1-GALR1 and UM-SCC-1-mock cells after galanin treatment. Pertussis toxin and LY294002 inhibition demonstrated that galanin and GALR1 induce ERK1/2 activation via Galphai, not the PI3K pathway-linked to the Gbetagamma subunit. Galanin and GALR1 also inhibit colony formation and tumor growth in vivo. Our results implicate GALR1, a Gi protein-coupled receptor, as a tumor suppressor gene that inhibits cell proliferation via ERK1/2 activation.