Receptor tyrosine kinase inhibition suppresses growth of pediatric renal tumor cells in vitro

PURPOSE: Children who undergo standard therapy for renal tumors are at an increased risk for treatment sequelae such as congestive heart failure, abnormal trunk development, and secondary malignancies. Therefore, research on the use of novel chemotherapeutic agents with fewer side effects is justified. Recent experimental evidence suggests that growth factor receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) play an important role in growth and development of pediatric renal tumors especially that of Wilms' tumor. In this study we investigated the effects of genistein, AG1478, and AG1295, from the class of growth factor receptor tyrosine kinase (GFR-TK) inhibitors, on proliferation and colonigenic growth of 2 pediatric renal tumor cell lines. METHODS: The authors studied the effect of genistein (broad-spectrum GFR-TK inhibitor), AG1478 (EGFR-specific GFR-TK inhibitor), and AG1295 (PDGFR-specific GFR-TK inhibitor) on proliferation and colonigenic growth of rhabdoid tumor of the kidney and Wilms' tumor cell lines: G-401 and SK-NEP-1, respectively. The effect of genistein at concentrations of 0 to 200 micromol/L, and AG1478 and AG1295 at 0 to 10,000 nmol/L were tested on proliferation by using a growth inhibition assay. Viable cell counts at each concentration were obtained by hemocytometer and trypan blue exclusion, and percent growth inhibition was calculated based on control cultures at the same time-point. As a measure of colonigenic survival, the percent inhibition of colony formation in drug-treated dishes was calculated based on the number of colonies (>50 cells) in control dishes. RESULTS: Genistein at concentrations of 25 and 50 micromol/L inhibited the colonigenic growth of G-401 by 37% and 79% (P = .01 and 5E-06, 2-tailed t test, respectively) and that of SK-NEP-1 by 44% and 74% (P = .0001 and 9.9E-07). The mean percent growth inhibition at the above doses was 57% +/- 7.9% and 96% +/- 0.2% for G-401, and 47% +/- 11.2% and 60% +/- 2.7% for SK-NEP-1. AG1478 at concentrations of 1,000 and 5,000 nmol/L inhibited the colonigenic growth of G401 by 75% and 78% (P = .0005 and 7.38E-06, respectively) and that of SK-NEP-1 by 19% and 40% (P = .02 and .0001). The percent growth inhibition at the mentioned concentrations for G-401 were 53% +/- 9.3% and 63% +/- 6.3%, and for SK-NEP-1 were 55% +/- 14.5% and 65% +/- 20.1%, respectively. AG1295 did not appear to be as effective as AG1478. CONCLUSIONS: This is the first experimental study on the use of GFR-TK inhibitors as a potential treatment for pediatric renal tumors. GFR-TK inhibitors such as genistein occur naturally in soybean foods and have been shown to reach therapeutic levels in blood after consuming a soybean-based diet. Considering the significance of growth factor receptor activity in Wilms' tumor development, inhibition of GFR-TKs should be investigated as effective and potentially nontoxic adjunctive treatment for this childhood tumor. Furthermore, GFR-TK inhibitors may offer an effective alternative to the treatment of commonly fatal rhabdoid tumor of the kidney in children.     

肿瘤干细胞与骨肉瘤相关研究进展

肿瘤研究中发现极少数有分化潜能,可无限增殖的亚细胞群具有干细胞特性,肿瘤的发生、生长、转移与之密切相关.肿瘤干细胞主要来源于成体干细胞,也可来源于祖细胞、分化细胞或骨髓细胞.白血病、乳腺癌、恶性脑瘤存在肿瘤干细胞已经明确,在前列腺癌、肺腺癌、肝癌、恶性黑色素瘤等多种肿瘤中也已初步分离出相应的肿瘤干细胞.骨肉瘤是常见的骨组织恶性肿瘤,肿瘤干细胞学说可更好地解释骨肉瘤恶性程度高、转移早、抗化疗等特性.应用无血清悬浮培养方法,干细胞样骨肉瘤细胞已初步得以分离、分析.该文就肿瘤干细胞及干细胞样骨肉瘤细胞的相关研究进展作一综述.     

蛋白酪氨酸磷酸酶-3通过诱导肿瘤性巨噬细胞钙离子依赖性钾离子通道表达增强LoVo细胞侵袭性

目的 观察结肠癌肿瘤微环境中肿瘤细胞与巨噬细胞(M2)相互作用及对肿瘤细胞功能学的改变.方法 将转入蛋白酪氨酸磷酸酶-3(PRL-3)的LoVo细胞和M2细胞模拟肿瘤微环境进行共培养,通过Western blot检测M2细胞钙离子依赖性钾离子通道(KCNN4)蛋白表达,并检测LoVo细胞侵袭性的改变.结果 Western blot检测示PRL-3能通过共培养后诱导M2细胞KCNN4蛋白表达升高,而未作共培养的M2细胞KCNN4蛋白表达未升高.此外,经过共培养后LoVo细胞侵袭性升高(3367±135比1442±89,P＜0.05),而在阻断KCNN4蛋白表达后,LoVo细胞侵袭性降低(1388 ±87比2893±163,P＜0.05).结论 PRL-3在结肠癌肿瘤微环境中通过提高KCNN4表达从而增强LoVo细胞的侵袭性.     

Protein tyrosine phosphatase 1 protects human pancreatic cancer from erastin-induced ferroptosis

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a fatal malignancy due to the lack of early detection method, therapeutic drug and target. We noticed that the expression of Protein Tyrosine Phosphatase Mitochondria1(PTPMT1) is upregulated in PDAC. However, its role in pancreatic cancer remains unknown.MethodsWe first analyzed the expression of PTPMT1 from 50 PDAC patients. Secondly, the survival proportions of different PTPMT1-expressed patients were analyzed. Then, the role and mechanism of PTPMT1 in PDAC were studied by lentivirus transduction system.ResultsPTPMT1 was upregulated in PDAC and patients with high PTPMT1 expression displayed lower overall survival rate. Knockdown of PTPMT1 increased the sensitivity to erastin or RSL3 induced ferroptosis. Mechanically, knockdown of PTPMT1 resulted in upregulated Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) and downregulated Solute Carrier Family 7 Member 11 (SLC7A11). In addition, SLC7A11 was upregulated in PDAC tumor tissue and correlated positively with the expression of PTPMT1. However, the expression of ACSL4 was downregulated in PDAC and negatively correlated with the expression of PTPMT1.ConclusionOur study demonstrates that PTPMT1 is upregulated in PDAC and PTPMT1 inhibits ferroptosis by suppressing the expression of ACSL4 and upregulating SLC7A11 in Panc-1 cells, suggesting PTPMT1 might be a potential prognosis biomarker and therapeutic target in PDAC.     

Protein tyrosine phosphatase PTP1B is involved in neuroendocrine differentiation of prostate cancer

BACKGROUND: Prostate cancer (PC) contains a minor component of neuroendocrine (NE) cells that may stimulate androgen-independent growth of the tumor. The mechanism of neuroendocrine differentiation remains unknown. METHODS: The expression of PTP1B, a protein tyrosine phosphatase, was studied in LNCaP cells induced to show neuroendocrine phenotype by androgen withdrawal. Wild-type PTP1B and its dominant-negative mutant were transfected into LNCaP cells to study their effects on neuroendocrine differentiation. In vivo expression of PTP1B in human prostate cancer was studied by immunohistochemistry. RESULTS: Androgen withdrawal of LNCaP cells led to increased expression of PTP1B with a corresponding increase in its tyrosine phosphatase activity. Overexpression of PTP1B in LNCaP cells led to neuroendocrine differentiation while expression of its dominant-negative mutant inhibited neuroendocrine differentiation. Immunohistochemical study showed that PTP1B was exclusively expressed in neuroendocrine cells of human prostate cancer tissue. CONCLUSION: Our findings suggest that PTP1B plays an important role in neuroendocrine differentiation, and therefore, may possibly be involved in the progression of prostate cancer.     

A phorbol ester induces secretion of alkaline phosphatase activity in human osteosarcoma cells

Summary The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) blocked the growth of, and induced the appearance of processes in the human osteosarcoma cell line U-2 OS. The phorbol ester decreased the intracellular level of alkaline phosphatase (APase) activity (as measured per mg cell protein) and caused a marked increase in the APase activity secreted from the cells into the culture medium. The secretion of APase appeared after a lag period of 4–6 hours of TPA treatment, and it could also be visualized with histological staining. Differential ultracentrifugation of the culture media showed that the APase was released to the media in the form of vesicles. The vesicles were studied by electron microscopy and appeared similar to matrix vesicles isolated from cartilage and chondrocytes. It is thus concluded that TPA is able to induce the primary steps of mineralization in these cells.     

Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis

Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor gamma. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85alpha) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity.     

新辅助化疗后肿瘤坏死率及碱性磷酸酶同工酶变化与预后的相关性研究

目的探讨骨肉瘤患者新辅助化疗后的肿瘤坏死率（TCNR）及碱性磷酸酶骨同工酶（BALP）变化与预后的关系。方法选取2003年1月至2007年3月初诊经活检病理诊断为骨肉瘤且排除转移的58例患者,术前常规新辅助化疗,根据对手术切除肿瘤标本的TCNR测定将患者分为两组（TCNR≥90％和〈90％）,分别随访两组患者的术后肺转移率和5年生存率并进行比较。同时检测患者在化疗前后及随访期间血清中BALP,并对检测结果进行分析。结果58例骨肉瘤患者中TCNR≥90％组为36例,〈90％组为22例,两组的术后肺转移率分别为16．7％和81．8％（x^2=12．156,P〈0．01）,5年生存率分别为72．2％和22．7％（x^2=8．125,P〈0．01）。化疗后BALP的下降比值与TCNR的升高呈正相关（r=0．735,P〈0．01）。随访期间肿瘤复发转移组与无瘤生存组BALP水平上差异有统计学意义（P〈0．01）。结论TCNR测定是评价骨肉瘤新辅助化疗反应较可靠方法,TCNR≥90％患者的远期转移率和生存时间明显优于TCNR〈90％患者。BALP是骨肉瘤血清学检查的高灵敏度指标,在骨肉瘤的治疗监测和预后的判断上有很高价值。TCNR和BALP两者同时检测可以提高预后精确性。     

Postoperative progression of pulmonary metastasis in osteosarcoma

Early relapse with distant metastasis often is observed in patients with cancer after resection of the primary tumor. It is considered that resection of the primary tumor induces activation of systemic angiogenesis and enhances progression of remote metastasis. The authors show that resection of the primary osteosarcoma tumor enhances progression of pulmonary metastasis in animal osteosarcoma models. Matrigel plug neovascularization assay revealed that systemic angiogenic activity was elevated after primary tumor removal (tumor intact group, 1.61 +/- 0.21 g/dL; tumor removed group, 4.92 +/- 0.35 g/dL). In addition, serum concentration of the angiogenesis inhibitor, endostatin, decreased significantly after primary tumor removal. Treatment with the antiangiogenic reagent TNP-470 suppressed postoperative progression of pulmonary metastasis. These results indicate the possibility that activation of angiogenic activity after resection of osteosarcoma tumors enhances progression of pulmonary metastasis. The current data also suggest that administration of antiangiogenic reagents can prevent progression of pulmonary metastasis in osteosarcoma postoperatively.     

Reversal of malignant phenotype in human osteosarcoma cells transduced with the alkaline phosphatase gene

Alkaline phosphatases are a family of glycoproteins that are able to hydrolize various monophosphate esters at a high pH optimum. Liver/bone/kidney (L/B/K) alkaline phosphatase (ALP) is one of the four major isoenzymes that belong to this family. Apart from its role in normal bone mineralization, other functions of L/B/K ALP remain obscure, both in physiological and in neoplastic conditions, including the bone-forming tumor osteosarcoma. In this study, we transfected the U-2 OS osteosarcoma cell line, which does not show any basal expression of this enzyme, with the full-length gene of L/B/K ALP, and analyzed the in vitro and in vivo features of four transfectants showing different expression of L/B/K ALP. A reduced in vitro ability to invade Matrigel and to grow in a semi-solid medium, together with a lower tumorigenic and metastatic ability in athymic mice, was found to be associated with a high level of cell surface L/B/K ALP activity. Moreover, L/B/K ALP transfectants showed a reduced secretion of matrix metalloproteinase-9 enzyme. These findings indicate a loss of aggressiveness of osteosarcoma cells after the expression of L/B/K ALP on their surface and suggest a new role for this enzyme.     

Association of protein tyrosine phosphatase 1B gene polymorphisms with type 2 diabetes

The PTPN1 gene codes for protein tyrosine phosphatase 1B (PTP1B) (EC 3.1.3.48), which negatively regulates insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase activation segment. PTPN1 is located in 20q13, a genomic region linked to type 2 diabetes in multiple genetic studies. Surveys of the gene have previously identified only a few uncommon coding single nucleotide polymorphisms (SNPs). We have carried out a detailed association analysis of 23 noncoding SNPs spanning the 161-kb genomic region, which includes the PTPN1 gene. These SNPs have been assessed for association with type 2 diabetes in two independently ascertained collections of Caucasian subjects with type 2 diabetes and two control groups. Association is observed between multiple SNPs and type 2 diabetes. The most consistent evidence for association occurred with SNPs spanning the 3' end of intron 1 of PTPN1 through intron 8 (P values ranging from 0.043 to 0.004 in one case-control set and 0.038-0.002 in a second case-control set). Analysis of the combined case-control data increased the evidence of SNP association with type 2 diabetes (P = 0.005-0.0016). All of the associated SNPs lie in a single 100-kb haplotype block that encompasses the PTPN1 gene. Analysis of haplotypes indicates a significant difference between haplotype frequencies in type 2 diabetes case and control subjects (P = 0.0035-0.0056), with one common haplotype (36%) contributing strongly to the evidence for association with type 2 diabetes. Odds ratios calculated from single SNP or haplotype data are in the proximity of 1.3. Haplotype-based calculation of population-attributable risk (PAR) results in an estimated PAR of 17-20% based on different models and assumptions. These results suggest that PTPN1 is a significant contributor to type 2 diabetes susceptibility in the Caucasian population. This risk is likely due to noncoding polymorphisms.     

Protein tyrosine kinase inhibitors block the stimulatory actions of phosphotyrosine phosphatase inhibitors to increase cell proliferation, alkaline phosphatase activity, and collagen synthesis in normal human bone cells

The present study sought to test whether inhibition of phosphotyrosine phosphatases (PTPs) would stimulate proliferation and differentiation of normal bone cells, and whether the PTP inhibitor-mediated effects would be blocked by protein tyrosine kinase (PTK) inhibitors. Three inhibitors [phenylarsine oxide (PAO), orthovanadate (VO(4)), and molybdate (MoO(4))] and two normal human bone cells with different basal differentiation status (i.e., mandible- and vertebra-derived bone cells) were used. Cell proliferation was determined with [(3)H]thymidine incorporation, and confirmed by cell counting. Bone cell differentiation was assessed by increases in alkaline phosphatase (ALP) specific activity and collagen synthesis. The three test PTP inhibitors each stimulated [(3)H]thymidine incorporation in both human bone cell types in a biphasic, dose-dependent manner with optimal doses of 20 nM PAO, 1 microM VO(4) and 2 microM MoO(4), respectively. These PTP inhibitors at mitogenic doses each significantly and reproducibly increased ALP specific activity and collagen synthesis. To determine whether the stimulatory effects of PTP inhibitors could be blocked by PTK inhibitors, the effects of tyrphostin A51 and erbstatin, two potent PTK inhibitors, on the actions of PTK inhibitors on [(3)H]thymidine incorporation and ALP specific activity were evaluated. Both tyrphostin A51 and erbstatin, which by themselves alone significantly inhibited human bone cell proliferation and increased ALP specific activity, completely abolished the stimulatory effects of each of the three test PTP inhibitors on bone cell proliferation and ALP specific activity. In conclusion, these findings confirm the premise that inhibition of PTP activities in normal human bone cells could lead to increases in cell proliferation and differentiation, effects that are independent of basal differentiation status of the cells. More importantly, this study demonstrates for the first time that the stimulatory actions of the PTP inhibitor on bone cell proliferation and ALP could be blocked by a PTK inhibitor, suggesting that the osteogenic effects of PTP inhibitors may depend on PTK activities, presumably to increase basal tyrosyl phosphorylation level. Accordingly, one should interpret results of studies using PTK inhibitors with caution in that an inhibition by a PTK inhibitor does not necessarily indicate the requirement of PTK activities, as it could also suggest involvement of an inhibition of PTPs.     

The tumor suppressive role of TIMP3 in the human osteosarcoma cells

BackgroundTissue inhibitor of metalloproteinase 3 (TIMP3) regulates a variety of cellular activities such as proliferation, viability, apoptosis, and motility. Functional loss of TIMP3 is reported in several human cancers. However, its role in osteosarcoma (OS) remains largely unclear.MethodsIn this study, we explored the mechanism underlying the modulation of TIMP3 in the growth and aggressiveness of U2OS and 143B human OS cells at both cellular and molecular levels.ResultsOur results show that overexpression of TIMP3 inhibits endogenous MMP activity and represses a series of oncogenic phenotypes of tumor cells independent of MMP inhibition, including reduced proliferation and survival, induced apoptosis, as well as improved sensitivity of tumor cells in response to cisplatin chemotherapy. TIMP3 overexpression also suppresses tumor cell invasion via its MMP inhibitory capacity. Importantly, TIMP3 modulates tumor cell oncogenesis via its induction of PTEN and subsequent inactivation of the PI3K/AKT pathway.ConclusionOur results suggest that TIMP3 is an oncosuppressor in human OS cells. Reactivation of TIMP3 function may be considered as a potential therapy for the treatment of this bone cancer.     

Effective osteosarcoma cytolysis using cytokine-induced killer cells pre-inoculated with tumor RNA-pulsed dendritic cells

Osteosarcoma with distant metastases at late stage has posed a challenge for novel therapeutic modalities. The application of cytokine-induced killer (CIK) cells to osteosarcoma constitutes a promising strategy. This approach had been studied in multiple myeloma and breast cancers, where CIK cells exhibited specific cytotoxicity toward malignant cells while sparing wild-type tissues. However, the consistency of CIK cell-induced anti-tumor cytotoxicity has not been thoroughly examined. We investigated whether autologous CIK cells could effectively induce cytolysis of cultured osteosarcoma cells. In addition to the observed CIK cell-induced osteosarcoma cytolysis, the pre-incubation of CIK cells with autologous dendritic cells pulsed with tumor's total RNA further enhanced the tumor cytolysis to greater than 6-fold. The anti-tumor cytolysis was optimized in complete autologous setting, and was attenuated with allogeneic components. The advantage of the co-culture with RNA-pulsed DC was lost when high CIK cell density was employed for anti-tumor cytotoxic assay, but was maintained in purified CD3(+)CD56(+) cells isolated from the CIK cells. This finding implied that CIK cells at limited cell density could induce effective osteosarcoma cytolysis with an aid from tumor antigen presentation on dendritic cell surface.     

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self‐apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self‐tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN‐α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid‐derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft‐infiltrating CD4⁺CD25⁺Foxp3⁺ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN‐α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.     

Osteotesticular protein tyrosine phosphatase expression in rodent testis

PURPOSE: In the last decade the novel receptor-like protein tyrosine phosphatase was identified and termed osteotesticular tyrosine phosphatase (OST-PTP) due to its restricted expression in bone and testis. OST-PTP expression is regulated during osteoblast differentiation and it shows stage specific expression in the testis. Confined OST-PTP expression in the basal compartment of the seminiferous tubules suggests that this protein may be a good candidate for a rodent germ stem cell marker. To test this hypothesis we determined the exact pattern of OST-PTP expression in the rodent testis. MATERIALS AND METHODS: Adult mouse and rat paraffinized testis sections were subjected to in situ hybridization using riboprobes against the receptor and catalytic domains of the protein OST-PTP. RESULTS: OST-PTP testicular expression in rodents is not confined to the spermatogonia, as previously suggested, but is also present in Sertoli cells in a stage independent pattern. This finding excludes the hypothesis that OST-PTP is a germ stem cell identification marker in rodents. In addition, we report the identification of a testicular OST-PTP isoform lacking part of a catalytic domain that is widely expressed during spermatogenesis in all cell types. CONCLUSIONS: This finding implies tight control over OST-PTP expression in the testis, which in turn suggests an important role for OST-PTP and its isoform in male germ cell differentiation.     

Genetic association between a lymphoid tyrosine phosphatase (PTPN22) and type 1 diabetes

The lymphoid-specific phosphatase (LYP) encoded by PTPN22 is involved in preventing spontaneous T-cell activation by dephosphorylating and inactivating T-cell receptor-associated Csk kinase. We have genotyped 396 type 1 diabetic patients and 1,178 control subjects of Caucasian descent from north central Florida and report a strong association between type 1 diabetes and a polymorphism (R620W) in the PTPN22 gene. The homozygous genotype for the T allele encoding the 620W residue is associated with an increased risk for developing type 1 diabetes (odds ratio [OR] = 3.4, P < 0.008), and the heterozygous genotype C/T had an OR of 1.7 (P = 6 x 10(-6)). The C/C homozygous genotype is protective against type 1 diabetes (OR = 0.5, P = 6 x 10(-6)). Furthermore, transmission disequilibrium analysis of 410 affected sibpair and simplex families of Caucasian descent indicated that the type 1 diabetes-associated T allele is transmitted more often (57.2%) than randomly expected (P < 0.003). Together with previous reports of the association between PTPN22 and type 1 diabetes, as well as rheumatoid arthritis and systemic lupus erythematosus, these results provide compelling evidence that LYP is a critical player in multiple autoimmune disorders.