Spiclomazine Induces Apoptosis Associated with the Suppression of Cell Viability,Migration and Invasion in Pancreatic Carcinoma Cells

The effective treatment for pancreatic carcinoma remains critically needed. Herein, this current study showed that spiclomazine treatment caused a reduction in viability in pancreatic carcinoma cell lines CFPAC-1 and MIA PaCa-2 in vitro. It was notable in this regard that, compared with pancreatic carcinoma cells, normal human embryonic kidney (HEK-293) and liver (HL-7702) cells were more resistant to the antigrowth effect of spiclomazine. Biochemically, spiclomazine treatment regulated the expression of protein levels in the apoptosis related pathways. Consistent with this effect, spiclomazine reduced the mitochondria membrane potential, elevated reactive oxygen species, and activated caspase-3/9. In addition, a key finding from this study was that spiclomazine suppressed migration and invasion of cancer cells through down-regulation of MMP-2/9. Collectively, the proposed studies did shed light on the antiproliferation effect of spiclomazine on pancreatic carcinoma cell lines, and further clarified the mechanisms that spiclomazine induced apoptosis associated with the suppression of migration and invasion.     

乳腺浸润性导管癌中PTTG与MMP-2和MMP-9 的表达及相关性

垂体瘤转化基因（PTTG）具有促进体外细胞转化和体内致瘤的能力,在乳腺癌组织中高表达,并与乳腺癌的复发和淋巴结转移有关.但PTTG 能否通过调节基质金属蛋白酶(MMP-2、MMP-9) 调控乳腺癌的侵袭与转移尚不清楚.本研究证明,PTTG可能因促进乳腺癌细胞中 MMP-2、MMP-9 分泌而在乳腺癌细胞侵袭、转移中发挥重要作用.免疫组织化学 PV 9000 通用型两步法显示,60例乳腺浸润性导管癌组织中,PTTG、MMP-2 和 MMP-9表达定位于肿瘤细胞胞浆,阳性率与周围正常乳腺组织相比,差异均具有统计学意义(P＜0.05).三者阳性表达均与淋巴结转移及TNM分期有关(P＜0.05);与患者年龄、肿瘤大小等无关(P＞0.05).乳腺浸润性导管癌中PTTG分别与MMP-2、MMP-9的表达呈正相关(P＜0.05).小RNA干扰技术干扰乳腺癌细胞株 MDA-MB-231 中的PTTG,Western印迹结果显示,干扰组与对照组相比,PTTG、MMP-2 和 MMP-9蛋白的表达水平明显下降. Transwell 侵袭实验显示,干扰组肿瘤细胞体外侵袭能力明显降低(P＜0.01).本研究表明,PTTG可能通过促进乳腺癌细胞中 MMP-2、MMP-9分泌,促进乳腺癌细胞的侵袭、转移.     

Integrated analysis of gene expression and methylation profiles of novel pancreatic cancer cell lines with highly metastatic activity

Pancreatic cancer is one of the most lethal human malignancies, partly because of its propensity for metastasis. However, highly metastatic human pancreatic cancer cell lines suitable for studies of metastasis are currently lacking. Here we established two highly metastatic human pancreatic cancer cell lines, MIA PaCa-2 In8 and Panc-1 In8, by Matrigel induction assay. The cell lines were further characterized both in vitro and in vivo. MIA PaCa-2 In8 and Panc-1 In8 cells demonstrated increased migration and invasion compared with their respective parental cells. Following injection into nude mice, MIA PaCa-2 In8 and Panc-1 In8 cells resulted in more pulmonary metastases compared with the parental cells. Furthermore, analyses of m RNA, long non-coding RNA, micro RNA, and methylation profiling revealed that these factors were aberrantly regulated in the highly metastatic cells,indicating that they probably affected metastasis. We thus established and characterized two highly metastatic human pancreatic cell lines that could be used as valuable tools for future investigations into the pathogenesis, metastasis, and potential treatment of human pancreatic cancer.     

特异性eIF-4EsiRNA重组腺病毒构建及其对人卵巢癌细胞SKOV-3转移相关能力的影响

目的：构建表达真核细胞起始因子-4E（eukaryoticinitiationfactor4E,eIF-4E）特异性siRNA的重组腺病毒载体,观察其对人卵巢癌细胞SKOV-3体外转移能力的影响。方法：应用基因重组技术将eIF．4EsiRNA序列构建于腺病毒载体pLP－Ade－X,经包装细胞包装后得到高滴度重组腺病毒pLP—Ade-4EsiRNA（psiE）。将腺病毒psiE感染SKOV．3细胞,用定量PCR进行elf．4E基因表达检测,然后应用transwell小室法观察对细胞侵袭和运动能力的影响,同时检测感染后细胞内VEGF、MMP－2、MMP－9蛋白表达。结果：病毒检测结果与预期相符,real—timePCR可检测到感染重组腺病毒psiE后SKOV．3细胞没有eIF-4E基因表达;病毒感染后transwell小室法检测到SKOV-3细胞的侵袭和运动能力均受到显著的抑制（均为P〈0．01）;此外病毒感染的SKOV．3细胞中VEGF、MMP-2、MMP-9蛋白表达降低。结论：封闭eIF-4E基因表达对人卵巢癌细胞SKOV．3的侵袭和运动都有抑制作用,其作用机制可能与VEGF、MMP-2、MMP-9表达相关。     

Berberine diminishes side population and down-regulates stem cell-associated genes in the pancreatic cancer cell lines PANC-1 and MIA PaCa-2

Cancer stem cells play an important role in metastasis and the relapse of drug resistant cancers. Side-population (SP) cells are capable of effluxing Hoechst 33342 dye and are referred to as cancer stem cells. We investigated the effect of berberine on pancreatic cancer stem cells of PANC-1 and MIA PaCa-2. For both cell lines, the proportions of SP cells in the presence of berberine were investigated and compared to the proportions in the presence of gemcitabine, a standard pancreatic anti-cancer drug. The proportions of SP cells in the PANC-1 and MIA PaCa-2 cell lines were about 9 and <0.1 %, respectively. After berberine and gemcitabine treatments, the SP cell proportion of PANC-1 decreased to 5.7 ± 2.0 and 6.8 ± 0.8 %, respectively, which compares to the control proportion of (9.7 ± 1.7). After berberine and gemcitabine treatment of PANC-1, of the four stem cell-associated genes (SOX2, POU5F1, NANOG, and NOTCH1), all but NOTCH1 were down-regulated. Unfortunately, the effect of berberine and gemcitabine treatments on MIA PaCa-2 SP cells could not be clearly observed because SP cells represented only a very small proportion of MIA PaCa-2 cells. However, SOX2, POU5F1, and NANOG genes were shown to be effectively down-regulated in the MIA PaCa-2 cell line as a whole. Taken together, these results indicate that berberine is as effective at targeting pancreatic cancer cell lines as gemcitabine. Therefore, we believe that POU5F1, SOX2, and NANOG can serve as potential markers, and berberine may be an effective anti-cancer agent when targeting human pancreatic cancer cells and/or their cancer stem cells.     

转hGM-CSF基因HepG2细胞疫苗侵袭性和生长活性初步检测

目的：探讨经60Co处理的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性变化。方法：体外培养三种肝癌细胞（①野生型HepG2肝癌细胞②转染hGM-CSF基因的HepG2肝癌细胞③60Co射线处理的转hGM-CSF基因的HepG2肝癌疫苗）采用MTT方法检测三种细胞在24h、48h、72h的OD值并绘出生长曲线;利用transwell小室进行体外侵袭实验来观察上述三种细胞侵袭性;用RT-PCR技术检测上述三种细胞基质金属蛋白酶2（MMP-2）在mRNA水平上表达的变化;结果：经60Co照射处理的转hGM-CSF基因的HepG2肝癌疫苗组OD值在相同培养时间点较其他两组明显变小且差异有显著性（P〈0.05）。三种细胞（上述①②③种细胞）transwell侵袭试验显示：③组穿过人造基底膜的细胞数量明显少于前两组;PT-PCR示：③组细胞的MMP-2的mRNA的表达明显低于①②。结论：经过60Co处理过的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性均明显降低。     

Stimulation of dorsal root ganglion neurons activity by pancreatic cancer cell lines

Stimulation of mice dorsal root ganglion neurons (DRGNs) activity by human pancreatic cancer (PanCa) cell line Mia PaCa-2 and its potential molecule mechanism has been investaged. DRGNs were cultured alone or along with the MIA PaCa-2. The effects of MIA PaCa-2 to DRGNs were determined by neurofilament (NF) immunocytochemical and Nissl staining. ELISA was used to detect the concentration of insulin-like growth factor-1 (IGF-1) in the culture supernatant. Cyton size, neurite outgrowth and neuronal activity in the experimental group were greater than in the control groups. However, the concentration of IGF-1 in the supernatants was not significantly different from those in the blank and non-cultured medium groups. In the presence of MIA PaCa-2 cell line, cyton size, neurite outgrowth and neuronal activity were enhanced, which may provide more routes for the invasion of cancer cells along nerves.     

沉默单核细胞趋化蛋白-1基因降低人食管癌EC109细胞的迁移及侵袭能力

单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)是白色脂肪细胞分泌的炎症趋化刺激因子,属于趋化因子CC亚族,可促进肿瘤血管形成和细胞外基质降解,从而促进肿瘤细胞的浸润与转移。沉默MCP-1基因可显著抑制恶性肿瘤生长及转移,但其作用的分子机制尚不完全清楚。本研究应用小干扰RNA技术沉默人食管癌EC109细胞中MCP-1表达。细胞划痕试验显示,与对照组相比,沉默MCP-1基因可明显抑制食管癌EC109细胞迁移能力。Transwell 侵袭实验显示,沉默MCP-1基因后,EC109细胞侵袭能力降低。Western 印迹试验和RT-PCR试验揭示,沉默MCP-1基因后,细胞中MMP-7、MMP-9、TGF-β1及VEGF表达水平显著下降。研究结果提示,沉默MCP-1基因可通过抑制MMP-7、MMP-9、TGF-β1及VEGF表达,降低癌细胞迁移及侵袭能力。     

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

Haishengsu (HSS) is a seashell protein extracted from Tegillarca L. granosa, a type of Malaysian shellfish. Previous in vitro studies showed that HSS might possess biological anticancer activity. In this combined in vitro and in vivo study, we investigated the inhibitory effects of HSS on tumor growth, invasion, and metastasis using human lung carcinoma cell lines A549 and NCI-H292, both intensely positive for matrix metalloproteinases-2 (MMP-2) and MMP-9. HSS significantly inhibited the proliferation of A549 and NCI-H292 as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The transwell chamber assay showed that HSS effectively blocked the invasion and migration of the carcinoma cells through the reconstituted extracellular matrix (Matrigel). Gelatin zymography analysis revealed that the secretion and activity of MMP-2 and MMP-9 in the supernatants of the cultured cells A549 and NCI-H292 were decreased after treatment with HSS. The levels of MMP-2 and MMP-9 in these cancer cells were further examined by Western blot assay in which a significant decrease of MMP-2 and MMP-9 was observed in A549 and NCI-H292 cells after 24 h of exposure to HSS. The anticancer activity of HSS was verified in a mouse model in which HSS delayed the growth of A549 xenografts after 3 weeks of oral administration. Inhibition of MMP-2 and MMP-9 expression was also demonstrated in the A549 xenografts as determined by Western blot analysis. These results suggest that HSS is a novel seashell protein that cannot only inhibit tumor growth but also prevent tumor invasion and metastasis through suppressing the activity of MMP-2 and MMP-9.     

siRNA联合沉默MMP-9和FAK基因对小鼠黑色素瘤高转移细胞B16F10体外侵袭和迁移的影响

目的： 观察双基因联合干扰MMP-9和FAK对小鼠黑色素瘤高转移细胞B16F10体外侵袭、迁移能力的影响。方法：分别构建pGV102-MMP9-siRNA,pGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,实验分为空白对照组、Anti-MMP-9组,Anti-FAK组、Anti-MMP-9 &FAK组、阴性对照组。经G418筛选GFP+克隆,流式细胞仪分析阳性率,激光共聚焦观察转染后细胞形态,半定量RT-PCR检测各组B16F10细胞MMP-9和FAK基因的mRNA转录水平,Transwell侵袭、迁移实验测定各组B16F10细胞体外侵袭、迁移能力。结果： 经G418筛选,3个转染组阳性率分别为92.41±1.64%,95.72±0.21%,91.52±0.11%,且转染后细胞形态良好;与空白对照组相比,3个转染组的MMP-9,FAK mRNA转录水平下降明显（P<0.01）,迁移、侵袭能力明显降低（P<0.01）,但Anti-MMP-9 &FAK组细胞侵袭迁移能力显著低于Anti-MMP-9 组和Anti-FAK组（P<0.01）。结论： 相比单独沉默MMP-9 或FAK,联合沉默MMP-9 和FAK可明显降低小鼠黑色素瘤B16F10细胞体外迁移、侵袭能力。     

The MAPK-activated protein kinase 2 mediates gemcitabine sensitivity in pancreatic cancer cells

Pancreatic carcinoma is the major clinical entity where the nucleoside analog gemcitabine is used for first-line therapy. Overcoming cellular resistance toward gemcitabine remains a major challenge in this context. This raises the need to identify factors that determine gemcitabine sensitivity in pancreatic carcinoma cells. We previously found the MAPK-activated protein kinase 2 (MK2), part of the p38/MK2 stress response pathway, to be required for DNA replication fork stalling when osteosarcoma-derived cells were treated with gemcitabine. As a consequence, inhibition or depletion of MK2 protects these cells from gemcitabine-induced death (Köpper, et al. Proc Natl Acad Sci USA 2013; 110:16856–61). Here, we addressed whether MK2 also determines the sensitivity of pancreatic cancer cells toward gemcitabine. We found that MK2 inhibition reduced the intensity of the DNA damage response and enhanced survival of the pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and Panc-1, which display a moderate to strong sensitivity to gemcitabine. In contrast, MK2 inhibition only weakly attenuated the DNA damage response intensity and did not enhance long-term survival in the gemcitabine-resistant cell line PaTu 8902. Importantly, in BxPC-3 and MIA PaCa-2 cells, inhibition of MK2 also rescued increased H2AX phosphorylation caused by inhibition of the checkpoint kinase Chk1 in the presence of gemcitabine. These results indicate that MK2 mediates gemcitabine efficacy in pancreatic cancer cells that respond to the drug, suggesting that the p38/MK2 pathway represents a determinant of the efficacy by that gemcitabine counteracts pancreatic cancer.     

α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as to cancer stemness features in pancreatic carcinoma

BackgroundPancreatic carcinoma is one of the deadliest malignant diseases, in which the increased expression of α1,6-fucosyltransferase (FUT8), a sole enzyme responsible for catalyzing core fucosylation, has been reported. However, its pathological roles and regulatory mechanisms remain largely unknown. Here, we use two pancreatic adenocarcinoma cell lines, MIA PaCa-2 and PANC-1 cells, as cell models, to explore the relationship of FUT8 with the malignant transformation of PDAC.MethodsFUT8 knockout (FUT8-KO) cells were established by the CRISPR/Cas9 system. Cell migration was analyzed by transwell and wound-healing assays. Cell proliferation was examined by MTT and colony-formation assays. Cancer stemness markers and spheroid formations were used to analyzed cancer stemness features.ResultsDeficiency of FUT8 inhibited cell migration and proliferation in both MIA PaCa-2 and PANC-1 cells compared with wild-type cells. Moreover, the expression levels of cancer stemness markers such as EpCAM, CXCR4, c-Met, and CD133 were decreased in the FUT8-KO cells compared with wild-type cells. Also, the spheroid formations in the KO cells were loose and unstable, which could be reversed by restoration with FUT8 gene in the KO cells. Additionally, FUT8-KO increased the chemosensitivity to gemcitabine, which is the first-line therapy for advanced pancreatic cancer.ConclusionsFUT8-KO reduced the cell proliferation and migration. Our results are the first to suggest that the expression of FUT8 is involved in regulating the stemness features of pancreatic cancer cells.General significanceFUT8 could provide novel insights for the treatment of pancreatic carcinoma.     

骨桥蛋白通过调控MMP-2和VEGF参与肝癌细胞侵袭的机制研究

目的：骨桥蛋白（Osteopontin,OPN）在肝癌细胞侵袭中的作用机制。方法：采用siRNA干涉的方法处理人肝癌细胞,用PCR和Western-blot法检测OPN的表达;用transwell小室检测不同处理后的HepG2和MHCC97H细胞的侵袭能力;采用Western．b1．ot和ELISA方法检测基质金属蛋白酶-2（matrixmetalloproteinase-2,MMP-2）和血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）蛋白表达和活力的变化情况。结果：在不同肝癌细胞系中,随着肝癌细胞系侵袭能力的增强,OPN的表达逐渐增高。siRNA可以降低HepG2和MHCC97H细胞中OPN的表达,并且能够降低HepG2和MHCC97H细胞的侵袭能力;抑制OPN的表达能够降低MMP-2和VEGF蛋白表达和蛋白活性。结论：OPN在肝癌侵袭过程中起着重要作用,其作用机制可能是通过调控MMP-2和VEGF蛋白表达和活性来参与肝癌的侵袭,OPN可作为肝癌侵袭转移治疗的新靶点。     

封闭EFNA2 基因表达对肝癌细胞HepG2 的转移和运动的抑制作用

目的:构建Ephrin-A2(EFNA2)基因的干扰RNA重组腺病毒,并观察其对肝癌细胞HepG2转移能力的影响。方法:合成EFNA2基因的干扰RNA片段,用基因重组技术构建于腺病毒载体pAD-X,经293细胞包装得到高滴度重组腺病毒pAEFNA2/siRNA,并用real-time PCR进行EFNA2基因表达的验证。将重组腺病毒pAEFNA2/siRNA感染HepG2细胞,48h后用transwell小室法检测病毒对HepG2细胞侵袭和运动能力的影响。结果:经验证病毒正确构建,且HepG2细胞感染病毒pAEFNA2/siRNA后48小时的real-time PCR检测结果未见到有EFNA2基因的表达。病毒感染后transwell小室可见,HepG2细胞的侵袭和运动能力均受到显著的抑制(分别为105±12 v.s.21±7cells/孔和194±22 v.s.39±11,P<0.01)。结论:成功构建了重组EFNA2siRNA腺病毒,并观察到封闭EFNA2对肝癌细胞HepG2的侵袭和运动均有抑制作用。     

Embelin Suppresses Growth of Human Pancreatic Cancer Xenografts,and Pancreatic Cancer Cells Isolated from KrasG12D Mice by Inhibiting Akt and Sonic Hedgehog Pathways

Pancreatic cancer is a deadly disease, and therefore effective treatment and/or prevention strategies are urgently needed. The objectives of this study were to examine the molecular mechanisms by which embelin inhibited human pancreatic cancer cell growth in vitro, and xenografts in Balb C nude mice, and pancreatic cancer cell growth isolated from Kras^G12D transgenic mice. XTT assays were performed to measure cell viability. AsPC-1 cells were injected subcutaneously into Balb c nude mice and treated with embelin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of Akt, and Sonic Hedgehog (Shh) and their target gene products were measured by the immunohistochemistry, and Western blot analysis. The effects of embelin on pancreatic cancer cells isolated from 10-months old Kras^G12D mice were also examined. Embelin inhibited cell viability in pancreatic cancer AsPC-1, PANC-1, MIA PaCa-2 and Hs 766T cell lines, and these inhibitory effects were blocked either by constitutively active Akt or Shh protein. Embelin-treated mice showed significant inhibition in tumor growth which was associated with reduced expression of markers of cell proliferation (Ki67, PCNA and Bcl-2) and cell cycle (cyclin D1, CDK2, and CDK6), and induction of apoptosis (activation of caspase-3 and cleavage of PARP, and increased expression of Bax). In addition, embelin inhibited the expression of markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9) in tumor tissues. Antitumor activity of embelin was associated with inhibition of Akt and Shh pathways in xenografts, and pancreatic cancer cells isolated from Kras^G12D mice. Furthermore, embelin also inhibited epithelial-to-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting the expression of Snail, Slug, and ZEB1. These data suggest that embelin can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt and Shh pathways, and can be developed for the treatment and/or prevention of pancreatic cancer.     

A cancer-specific anti-podocalyxin monoclonal antibody (60-mG2a-f) exerts antitumor effects in mouse xenograft models of pancreatic carcinoma

Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy to target PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb (CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into a mouse IgG_2a-type mAb, named as 60-mG_2a, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed a core fucose-deficient type of 60-mG_2a, named as 60-mG_2a-f, to augment its ADCC activity. In vivo analysis revealed that 60-mG_2a-f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/mouse/week administered three times. These results suggested that 60-mG_2a-f could be useful for antibody-based therapy against PODXL-expressing pancreatic cancers.     

Effects of inhalation anesthetics halothane, sevoflurane, and isoflurane on human cell lines

Cytotoxic and antiproliferative effects of halothane, isoflurane, and sevoflurane in anesthetic doses on human colon carcinoma (Caco-2), larynx carcinoma (HEp-2), pancreatic carcinoma cells (MIA PaCa-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), and normal fibroblasts were investigated. Cells were exposed to anesthetic gas mixture consisting of O(2): N2O (35:60 vol.%), halothane (1.5 vol.%) or isoflurane (2.0 vol.%) or sevoflurane (3.0 vol.%), and CO(2) (5 vol.%), for 2, 4, and 6 h. Cytotoxicity of anesthetics was analyzed by validated tetrazolium dye assay MTT test. All anesthetics expressed cytotoxic effects on treated tumor cells in time and cell line dependent manner. Growth suppression in cells exposed to halothane was enhanced in HEp-2 (to 67.7%), Caco-2 (to 76.3%), and SW620 cells (to 80.9%), and was minimal in normal fibroblasts (to 89.4%). Antiproliferative activity of halothane was measured via radioactive precursors incorporation assay. In Caco-2 cells treated by halothane, decrease in DNA synthesis (52.4%, p=0.001), RNA synthesis (39.2%, p<0.001), and protein synthesis (19.2%, p=0.004) was observed. In HEp-2 cells, DNA and RNA syntheses were decreased to 72.5% and 79.9%, whereas protein synthesis was 14.0% of control (p<0.001). In SW620 cells, protein synthesis after 4 h was 24.4% (p=0.007). A DNA fragmentation was observed in Caco-2 and MIA PaCa-2 cells. Exposition of phosphatidylserine on outer lipid bilayer plasma membrane of tumor cell treated by halothane proved apoptosis as mode of cell death.     

CXCR4 promotes oral squamous cell carcinoma migration and invasion through inducing expression of MMP-9 and MMP-13 via the ERK signaling pathway

The increased migration and invasion of oral squamous cell carcinoma cells are key events in the development of metastasis to the lymph nodes and distant organs. Although the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1α, have been found to play an important role in tumor invasion, its precise role and potential underlying mechanisms remain largely unknown. In this study, we showed that knockdown of CXCR4 significantly decreased Tca8113 cells migration and invasion, accompanied with the reduction of MMP-9 and MMP-13 expression. Inhibition of ligand binding to CXCR4 by a specific antagonist TN14003, also led to reduced cancer cell migration and invasion. Because the degradation of the extracellular matrix and the basement membrane by proteases, such as matrix metalloproteinases (MMP) is critical for migration and invasion of cancer cells, we investigated the expression of several MMPs and found that the expression of functional MMP-9 and MMP-13 was selectively decreased in CXCR4 knockdown cells. More importantly, decreased cell migration and invasion of CXCR4 knockdown cells were completely rescued by exogenous expression of MMP-9 or MMP-13, indicating that the two MMPs are downstream targets of CXCR4-mediated signaling. Furthermore, we found the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in CXCR4-silenced cells, suggesting that ERK may be a potential mediator of CXCR4-regulated MMP-9 and MMP-13 expression in Tca8113 cells. Taken together, our results strongly suggest the underlying mechanism of CXCR4 promoting Tca8113 migration and invasion by regulating MMP-9 and MMP-13 expression perhaps via activation of the ERK signaling pathway.