Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells

The human tissue kallikrein family of serine proteases (hK1-hK15 encoded by the genes KLK1-KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the 'tissue kallikrein overexpressing' group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; chi2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.     

Kallikrein 6 is a mediator of K-RAS-dependent migration of colon carcinoma cells

Kallikrein 6 (KLK6) is a trypsin-like serine peptidase whose relevance in various types of cancers is currently being explored. Previous studies have shown that KLK6 mRNA is upregulated in colon and gastric cancers; however, the regulatory mechanisms and phenotypic consequences of this upregulation are largely unknown. Activating K-RAS mutations are common in colon cancer, occurring in approximately 50% of cases. We have recently reported the upregulation of KLK6 mRNA in Caco2 human colon cancer cells stably transfected with a mutant K-RAS allele (K-RAS(G12V)). In this study we examined the pattern of K-RAS-dependent KLK6 expression and secretion in colon cancer cells. Using pharmacological inhibitors of pathways downstream of K-RAS, we could show that the PI3K and p42/44 MAPK pathways play an important role in the induction of KLK6 in mutant K-RAS-expressing colon cancer cells. Increased KLK6 expression enhanced colon cancer cell migration through laminin and Matrigel. Inhibition of KLK6 using small interference RNA treatment or a specific KLK6 antibody in Caco2 cells stably expressing the mutant K-RAS and in SW480 cells carrying a mutation in the K-RAS oncogene resulted in a reduction in invasiveness through cell culture inserts. These data support the oncogenic role of KLK6 in colorectal cancer.     

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

Gliomas are the most lethal tumors of central nervous system. ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor ligand at high concentrations. Herein, we investigated whether the activation of P2X7R might be implicated in death of a radiosensitive human glioma lineage. The effects of P2X7R agonists (ATP and BzATP) and irradiation (2 Gy) on glioma cells were analyzed by MTT assay and annexin-V/PI determination, whereas mRNA and protein P2X7R expression was assessed by qRT-PCR and flow cytometry, respectively. P2X7R pore formation was functionality examined by analyzing ethidium bromide uptake. The human glioma cells U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), but the radiosensitive M059J glioma cells displayed a significant decrease of cell viability (32.4 ± 4.1 % and 25.6 ± 3.3 %, respectively). The M059J lineage expresses significantly higher mRNA P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40 ± 0.00; 0.28 ± 0.01, and 0.31 ± 0.01, respectively), and irradiation upregulated P2X7R expression (0.55 ± 0.08) in this lineage. Noteworthy, P2X7R protein doubled after irradiation on M059J lineage, and increased in 50 % and 42.6 % when comparing M059J-irradiated to irradiated U-138 MG and U-251 MG cells, respectively. Ethidium bromide uptake was significantly increased in 104 % and 77.8 % when comparing M059J to U-138 MG and U-251MG, respectively. Finally, the selective P2X7R antagonist A740003 significantly decreased the cell death caused by irradiation. We provide novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R on glioma radiosensitivity.     

Kallikrein 1 is overexpressed by astrocytes in the hippocampus of patients with refractory temporal lobe epilepsy, associated with hippocampal sclerosis

Kallikrein 1 (hK1) is a tissue enzyme responsible for kinin release in inflammatory cascade. This study was delineated to study the distribution and the co-localization of hK1 and kinin B1 and B2 receptors with glial and/or neuronal proteins markers, in the hippocampus of patients with refractory temporal lobe epilepsy, associated with hippocampal sclerosis (TLE-HS), comparing with control tissues. Hippocampal levels of KLK1 mRNA were also measured. hK1, kinin B1 and B2 receptors, NeuN and GFAP were analyzed using immunohistochemistry and confocal microscopy and KLK1 mRNA was quantified with real time PCR. Increased expression of hK1 by astrocytes co-localized with GFAP was found, contrasting with kinin B1 and B2 receptors, which were co-localized with NeuN in the sclerotic hippocampus. In addition, KLK1 mRNA was also up-regulated in same tissues. These data suggest an overexpression of kallikrein-kinin system and a neuron-glia interaction in the inflammatory process present in refractory TLE-HS.     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Human tissue kallikrein 9: production of recombinant proteins and specific antibodies

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.     

The kallikrein 14 gene is down-regulated by androgen receptor signalling and harbours genetic variation that is associated with prostate tumour aggressiveness

Kallikrein 14 (KLK14) has been proposed as a useful prognostic marker in prostate cancer, with expression reported to be associated with tumour characteristics such as higher stage and Gleason score. KLK14 tumour expression has also shown the potential to predict prostate cancer patients at risk of disease recurrence after radical prostatectomy. The KLKs are a remarkably hormone-responsive family of genes, although detailed studies of androgen regulation of KLK14 in prostate cancer have not been undertaken to date. Using in vitro studies, we have demonstrated that unlike many other prostatic KLK genes that are strictly androgen responsive, KLK14 is more broadly expressed and inversely androgen regulated in prostate cancer cells. Given these results and evidence that KLK14 may play a role in prostate cancer prognosis, we also investigated whether common genetic variants in the KLK14 locus are associated with risk and/or aggressiveness of prostate cancer in approximately 1200 prostate cancer cases and 1300 male controls. Of 41 single nucleotide polymorphisms assessed, three were associated with higher Gleason score (≥7): rs17728459 and rs4802765, both located upstream of KLK14, and rs35287116, which encodes a p.Gln33Arg substitution in the KLK14 signal peptide region. Our findings provide further support for KLK14 as a marker of prognosis in prostate cancer.     

KLK5 and KLK7, two members of the human tissue kallikrein family, are differentially expressed in lung cancer

Emerging data indicate that serine proteases of the kallikrein family (KLK) are implicated in various human diseases, including carcinoma; however, kallikrein gene expression has never been investigated in lung cancer. Using RT-PCR and Western blotting, we demonstrated the expression of both KLK5 and KLK7, and their respective proteins (hK5 and hK7) in tumoral and nontumoral lung tissues. Quantitative gene expression was then analyzed in a cohort of 56 patients with non-small cell lung cancer by real-time RT-PCR. KLK5 expression is significantly more expressed in squamous cell carcinoma than in matched nonmalignant lung tissue (P=0.02), whereas expression of KLK7 was decreased in adenocarcinoma (P=0.003). Multivariate analysis revealed diverse correlations between the KLK5 and KLK7 expression levels in nonmalignant and malignant tissues, and clinical parameters, including histotype, metastatic status, and grade. Our findings provide new insight into kallikrein gene expression in hormone-independent carcinoma. Altogether, our results suggest that variability in KLK5 and KLK7 gene expression might be involved in lung tumorigenesis and useful for clinical purposes.     

Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.     

基于网络药理学分析瑞香素抗多种肿瘤的作用机制及体外实验验证

基于网络药理学预测瑞香素抗恶性胶质瘤、肝癌和三阴性乳腺癌的共同靶点及可能机制,并对其进行体外实验验证。利用Swiss Target Prediction和GeneCards等数据库检索瑞香素与恶性胶质瘤、肝癌和三阴性乳腺癌的共同靶点。使用Cytoscape构建瑞香素-三种肿瘤蛋白质相互作用网络图(PPI)并筛选出核心靶点,并对核心靶点进行GO及KEGG富集分析;通过AutoDock Tools对瑞香素与核心靶点进行分子对接。体外实验验证:采用CCK-8和Western blot法行体外实验验证不同浓度瑞香素对U-251 MG、HepG-2和MDA-MB231细胞系细胞抑制率和P53、RRM2蛋白的表达水平的影响。共筛选出瑞香素抗三种肿瘤核心靶点56个,富集分析显示靶点富集在P53通路和癌症通路,参与细胞周期调节、细胞凋亡、DNA生物合成和修复等生物过程;分子对接结果显示瑞香素与P53、RRM2有较好的结合作用。体外验证实验显示,与对照组比较,瑞香素能显著抑制U-251 MG、HepG-2、MDA-MB231的增殖(P<0.01),并显著上调其P53蛋白及下调RRM2蛋白的表达,且...     

Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells

Impaired function of apoptosis-related genes is deeply involved in oncogenesis and the progression of cancers, and caspase-3 plays a critical role as an executioner of apoptosis. We introduced the caspase-3 gene via an adenovirus (Adv) vector into Alexander hepatoma cells, MCF-7 breast cancer cells, and U251 and U-373MG glioma cells which have different endogenous levels of caspase-3 expression. None of the cell lines underwent apoptosis by overexpression of caspase-3, indicating that induction of caspase-3 alone is not applicable for cancer gene therapy. Next, we investigated whether overexpression of caspase-3 could enhance Fas ligand-mediated apoptosis in these four cell lines. In U-373MG cells, which showed the highest level of expression of surface Fas among the four cell lines, coinfection of the Adv for caspase-3 (Adv-caspase-3) and the Adv for Fas ligand (Adv-FL) induced a remarkably increased degree of apoptosis compared with that induced by the single infection of either Adv-caspase-3 or Adv-FL. Similar results were obtained by cotreatment with anti-Fas antibody in U-373MG cells. These data suggest that when strong proapoptotic upstream stimuli are induced, the level of caspase-3 expression determines the degree of apoptosis in cancer cell lines. In conclusion, overexpression of caspase-3 alone did not induce apoptosis in cancer cells. Both a strong proapoptotic signal and a high expression of caspase-3 were required to induce drastic apoptosis in cancers. This strategy would be highly beneficial for selected cancer patients.     

Quantitative RT-PCR analysis and immunohistochemical localization of the kallikrein-related peptidases 13 and 14 in lung

Expression of the KLK13 and KLK14 genes was examined at the mRNA and protein levels in a cohort of 57 patients with non-small-cell lung cancer (NSCLC). The mRNA levels, assessed by real-time RT-PCR, were significantly different in malignant tissues compared to adjacent non-malignant tissues (KLK13, p=0.006; KLK14, p=0.022). KLK13 and KLK14 mRNA overexpression in tumors (1/3 of the patients) was associated with a positive nodal status in multivariate analysis (p=0.018 and p=0.069, respectively). KLK13 and KLK14 were localized in the cytoplasm of epithelial cells of normal bronchus and NSCLC, as determined by immunohistochemistry. Moreover, positive staining was significantly associated with adenocarcinoma histotype (KLK13, p=0.014) and tumor size (KLK14, p=0.048). Although the results are marginally significant, patients with high KLK13 expression at the mRNA or protein level had lower overall survival.     

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.     

Adenovirus-mediated transfer of caspase-8 in combination with superrepressor of NF-kappaB drastically induced apoptosis in gliomas

Inhibition of NF-kappaB in the presence of tumor necrosis factor-alpha (TNF) is supposed to be a promising cancer therapeutic approach, since it disrupts the protective mechanism of NF-kappaB activated by TNF. To test this approach in gliomas, we introduced a superrepressor of NF-kappaB, an N-terminal deleted form of inhibitor kappa B alpha (IkappaBdN) gene, to human glioma cells (U251 and U-373MG) via adenoviral vector (Adv) in the presence of TNF. U-373MG cells were refractory to TNF-induced apoptosis even when they were transduced with the IkappaBdN gene. On the other hand, transduction of IkappaBdN drastically augmented caspase-8-mediated apoptosis in U-373MG cells. Similar results were obtained in U251 cells. Cotransduction of IkappaBdN and caspase-8 induced cleavage of PARP. Taken together, Adv-mediated transfer of IkappaBdN plus caspase-8 may be a promising therapeutic approach to treat gliomas.     

The extracellular matrix protein fibronectin is a substrate for kallikrein 7

Kallikrein 7 (hK7), a chymostatin-like serine protease, is overexpressed in pancreatic adenocarcinomas as well as other human cancers. Although it has been demonstrated to participate in normal desquamation by facilitating cell shedding at the skin surface, its role in human malignancies remains unclear. To investigate the ability of hK7 to degrade components of the extracellular matrix (ECM), recombinant hK7 was expressed and purified from cultured mammalian cells. Using a three-step chromatographic purification procedure, recombinant hK7 was obtained that displayed robust proteolytic activity against a fluorogenic peptide substrate following activation by thermolysin. We demonstrate that the active protease is able to cleave fibronectin in a time-dependent manner, but not laminin, using an in vitro degradation assay. These findings indicate that the aberrant expression and secretion of hK7 in human tumors may facilitate metastasis by directly degrading components of the extracellular matrix and may thus play an important role in tumorigenesis.