c-Met Overexpression Contributes to the Acquired Apoptotic Resistance of Nonadherent Ovarian Cancer Cells through a Cross Talk Mediated by Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/2

Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications.     

Cisplatin-induced cytotoxicity is blocked by brain-derived neurotrophic factor activation of TrkB signal transduction path in neuroblastoma

We evaluated the ability of brain-derived neurotrophic factor (BDNF) to decrease the chemosensitivity of neuroblastoma cells to cisplatin. Two cell lines, one derived from SH-SY5Y (SY5Y-TB8) and the other from SK-N-AS (AS-TB8), transfected with a TrkB plasmid were generated, and used to assess the effects of activation of the TrkB signal transduction path on cisplatin (Cis) induced apoptosis. BDNF treatment of each of the TrkB expressing cells blocked cisplatin-induced cell death. BDNF's ability to rescue the cells from cisplatin-induced cell death was inhibited by treatment with the Trk tyrosine kinase inhibitor, K252a, and the phosphatidylinositol 3'-kinase (PI)-3-kinase inhibitor, LY294002. This indicates that the activation of the TrkB path through PI-3-kinase is required for BDNF's survival-promoting effects.     

卵巢癌多细胞球体对紫杉醇耐药及其机制的探讨

背景和目的:多细胞球体(multicellularspheroids,MCS)对传统细胞毒化疗药物的敏感性明显低于单层细胞。本实验旨在探讨卵巢癌MCS耐药的分子机制。方法:以单层细胞为对照,以三维培养方法获得的人卵巢癌A2780MSC和CAOV3MCS为模型,采用台盼蓝拒染法比较紫杉醇对单层细胞和MCS生长的抑制作用,流式细胞仪比较单层细胞和MCS细胞周期的分布和细胞凋亡率;采用流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测单层细胞及MCS的P-糖蛋白(P-glycoprotein,P-gp)表达及亚细胞分布;采用RT-PCR法检测mdr1mRNA表达水平。结果:(1)不同浓度的紫杉醇(0.2、2.0、10.0、20.0μmol/L)作用后,MCS细胞生长抑制率明显低于单层细胞(PA2780=0.003,PCAOV3=0.015);经20.0μmol/L的紫杉醇作用后,单层细胞的细胞凋亡率明显高于MCS,差异有统计学意义(PA2780=0.034,PCAOV3=0.032)。(2)流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测提示P-gp在单层细胞中不表达,在MCS中表达明显升高(P均<0.05);RT-PCR证实MCS中有mdr1mRNA表达,而单层细胞中未检出其表达。(3)流式细胞仪检测提示将单层细胞培养成MCS时,G0/G1期细胞比率增加,S期和G2/M期细胞比率降低(P均<0.05)。结论:卵巢癌MCS对紫杉醇化疗耐药性增加,其高表达P-gp,并且与G0/G1期细     

Brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from chemotherapy-induced apoptosis via phosphatidylinositol 3'-kinase pathway

Neuroblastoma (NB) tumors expressing high levels of brain-derived neurotrophic factor (BDNF) and TrkB are associated with poor 5-year survival outcomes. Our previous studies indicated that BDNF blocked the cytotoxic effects of vinblastine on NB cells. Here we evaluated the ability of BDNF to decrease the chemosensitivity of NB cells to a number of common chemotherapeutic agents. Two SH-SY5Y NB cell lines (TB3 and TB8) expressing TrkB under the control of a tetracycline (Tet)-repressible promoter element were generated, and used to assess apoptosis resulting from treatment with cisplatin, doxorubicin, etoposide, and vinblastine. BDNF treatment of high TrkB-expressing TB8 (Tet-) and TB3 (Tet-) cells blocked drug-induced cell death in a dose-dependent manner. Only high-dose BDNF (100 ng/ml) could block the effects of chemotherapy in low TrkB-expressing cells. The ability of BDNF to rescue the cells from chemotherapeutic agent-induced cell death was inhibited by treatment with the Trk tyrosine kinase inhibitor K252a or the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not by the mitogen-activated protein kinase kinase inhibitor PD98059 or the peritoneal lymphocyte gamma inhibitor U73122, indicating that both TrkB and PI3K activities are required for the survival-promoting effects of BDNF. BDNF also protected TrkB-expressing NGP and KCNR NB cells from chemotherapeutic agent-induced cell death, and LY294002 inhibited this protection. These results suggest that TrkB and BDNF can contribute to the chemoresistance of poor prognosis tumors, and that suppression of PI3K activity might improve the ability of these agents to induce the death of NB tumors.     

In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002).

Phosphatidylinositol 3-kinase (PI3-K) induces mitogenesis, cell growth, and cell transformation. Amplification of the gene encoding the P110alpha subunit likely is an important event in ovarian cancer progression, and PI3-K inhibitors are possible therapeutic agents for this disease. We evaluated effects of LY294002, a potent inhibitor of PI3-K, on growth of ovarian carcinoma in vivo and in vitro, and on ascites formation in vivo. Athymic mice were inoculated i.p. with the ovarian cancer cell line OVCAR-3. Seven days after inoculation, mice were treated with or without LY294002 (100 mg/kg of body weight) for 3 weeks. Body weight and abdominal circumference were measured twice weekly. At the end of the experiment, mice were sacrificed, ascites volume was measured, and tumors were excised. Mean tumor burden in the LY294002-treated group was reduced by approximately 65% versus controls. Virtually no ascites developed in the treatment group; mean volume of ascites in controls was 3.3 +/- 0.38 ml. OVCAR-3 cells also were cultured in vitro without and with LY294002 (1, 5, and 10 microM) for 24 h. The number of cells in 1, 5, and 10 microM LY294002-treated wells was reduced by 27, 56, and 75%, respectively, versus controls. In vivo and in vitro morphological studies demonstrated that LY294002 induced marked nuclear pyknosis and diminished cytoplasmic volume in the tumor cells, confirmed as apoptosis. Thus, LY294002 significantly inhibits growth and ascites formation of ovarian carcinoma in vivo and markedly inhibits ovarian cancer cell proliferation in vitro, suggesting an important role of PI3-K inhibitors as a potentially useful treatment for women with ovarian carcinoma.     

E-cadherin cell-cell adhesion in ewing tumor cells mediates suppression of anoikis through activation of the ErbB4 tyrosine kinase

Ability to grow under anchorage-independent conditions is one of the major hallmarks of transformed cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To model this phenomenon in vitro, we plated Ewing tumor cells under anchorage-independent conditions by transferring them to dishes coated with agar to prevent attachment to underlying plastic. This resulted in marked up-regulation of E-cadherin and rapid formation of multicellular spheroids in suspension. Addition of calcium chelators, antibodies to E-cadherin (but not to other cadherins or beta(1)-integrin), or expression of dominant negative E-cadherin led to massive apoptosis of spheroid cultures whereas adherent cultures were unaffected. This correlated with reduced activation of the phosphatidylinositol 3-kinase-Akt pathway but not the Ras-extracellular signal-regulated kinase 1/2 cascade. Furthermore, spheroid cultures showed profound chemoresistance to multiple cytotoxic agents compared with adherent cultures, which could be reversed by alpha-E-cadherin antibodies or dominant negative E-cadherin. In a screen for potential downstream effectors of spheroid cell survival, we detected E-cadherin-dependent activation of the ErbB4 receptor tyrosine kinase but not of other ErbB family members. Reduction of ErbB4 levels by RNA interference blocked Akt activation and spheroid cell survival and restored chemosensitivity to Ewing sarcoma spheroids. Our results indicate that anchorage-independent Ewing sarcoma cells suppress anoikis through a pathway involving E-cadherin cell-cell adhesion, which leads to ErbB4 activation of the phosphatidylinositol 3-kinase-Akt pathway, and that this is associated with increased resistance of cells to cytotoxic agents.     

SMYD3 promotes implant metastasis of ovarian cancer via H3K4 trimethylation of integrin promoters

Detachment of cancer cells from the primary tumor and formation of spheroids in ascites is required for implantation metastasis in epithelial ovarian cancer (EOC), but the underlying mechanism of this process has not been thoroughly elucidated. To mimic this process, ovarian cancer cells were grown in 3D and 2D culture. Hey and OVCA433 spheroids exhibited decreased cell proliferation and enhanced adhesion and invasion. SMYD3 expression was elevated in ovarian carcinoma spheroids in association with increased H3K4 methylation. Depletion of SMYD3 by transient siRNA, stable shRNA knockdown and the SMYD3 inhibitor BCI-121 all decreased spheroid invasion and adhesion. Gene expression arrays revealed downregulation of integrin family members. Inhibition assays confirmed that invasion and adhesion of spheroids are mediated by ITGB6 and ITGAM. SMYD3-deficient cells regained the ability to invade and adhere after forced overexpression of SMYD3, ITGB6 and ITGAM. However, this biological ability was not restored by forced overexpression of SMYD3 in ITGB6- and/or ITGAM-deficient cancer cells. SMYD3 and H3K4me3 binding at the ITGB6 and ITGAM promoters was increased in spheroids compared to that in monolayer cells, and the binding was decreased when SMYD3 expression was inhibited, consistent with the expression changes in integrins. SMYD3 expression and integrin-mediated adhesion were also activated in an intraperitoneal xenograft model and in EOC patient spheroids. In vivo, SMYD3 knockdown inhibited tumor metastasis and reduced ascites volume in both the intraperitoneal xenograft model and a PDX model. Overall, our results suggest that the SMYD3-H3K4me3-integrin pathway plays a crucial role in ovarian cancer metastasis to the peritoneal surface.     

SOX2 is required to maintain cancer stem cells in ovarian cancer

Ovarian cancer cells can form spheroids under serum‐free suspension culture conditions. The spheroids, which are enriched in cancer stem cells, can result in tumor dissemination and relapse. To identify new targetable molecules in ovarian cancer spheroids, we investigated the differential expression of genes in spheroids compared with that under monolayer culture conditions by qPCR microarray. We identified that SOX2 is overexpressed in spheroids. We then proved that SOX2 expression was increased in successive spheroid generations. Besides, knockdown of SOX2 expression in SKOV3 or HO8910 ovarian cancer spheroid cells decreased spheroid formation, cell proliferation, cell migration, resistance to Cisplatin treatment, tumorigenicity, and the expression of stemness‐related genes and epithelial to mesenchymal transition‐related genes, whereas overexpression of SOX2 in SKOV3 or HO8910 ovarian cancer cells showed the opposite effects. In addition, we found that SOX2 expression was closely associated with chemo‐resistance and poor prognosis in EOC patients. These results strongly suggest that SOX2 is required to maintain cancer stem cells in ovarian cancer. Targeting SOX2 in ovarian cancer may be therapeutically beneficial.     

Critical role for TrkB kinase function in anoikis suppression, tumorigenesis, and metastasis

Anoikis, or cell death induced by cell detachment, provides protection against the metastatic spread of tumor cells. We have previously shown that the neurotrophic receptor tyrosine kinase TrkB suppresses anoikis in rat intestinal epithelial cells and renders them highly tumorigenic and metastatic. Because TrkB is overexpressed in several aggressive human cancers, first attempts are being made to target TrkB in cancer therapy. However, the mechanisms underlying TrkB-mediated anoikis suppression, tumorigenesis, and metastasis still remain largely elusive. Although, to date, most attempts to neutralize TrkB in tumors aim to inactivate its kinase activity, it is unclear whether TrkB kinase activity is required for its oncogenic functions. Indeed, it has been suggested that also other properties of the receptor contribute to functions that are relevant to tumor cell survival. Specifically, several adhesion motifs reside within the extracellular domains of TrkB. In line with this, TrkB-expressing epithelial cells form large cellular aggregates in suspension cultures, possibly facilitating tumor cell survival. Therefore, we set out to study the relative contributions of TrkB's kinase activity and its adhesion domains to anoikis suppression and oncogenicity. On the basis of a structure-function analysis, we report that TrkB kinase activity is required and, unexpectedly, also sufficient for anoikis suppression, tumor formation, and experimental metastasis. Thus, TrkB can act tumorigenically independent of its adhesion motifs. These results suggest that targeting the enzymatic activity of TrkB might be beneficial in cancer therapy.     

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/αv integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and αv integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and αv integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-αv integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/αv integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.     

Oncolysis of human ovarian cancers by echovirus type 1

A small number of enteroviruses possess the capacity to induce rapid and marked lytic infections in cells of various human malignancies. During screening of representative human enteroviruses for their oncolytic capacity, we observed that echovirus type 1 (EV1) displayed a high level of tropism for human ovarian cancer cells. EV1 is an enterovirus which largely causes asymptomatic infections in humans and whose tissue tropism is primarily regulated via interactions with the I domain of the alpha subunit of cell surface-expressed integrin alpha2beta1. We evaluated the capacity of wild-type EV1 to act as an oncolytic agent of ovarian cancers propagated as cell monolayers, multicellular spheroids or xenografts in SCID mice. EV1 infection of in vitro propagated ovarian cell lines expressing high levels of integrin alpha2beta1 was assessed for specific viral attachment, antibody blockade, induction of cytopathic effect and production of progeny virions. EV1 lytically infected all 8 human ovarian cancer cell lines tested (2008, DOV13, JAM, OVCA-429, OVCAR-3, OVHS-1, OAW-42 and IGROV-1) but not the immortalized normal ovarian surface epithelial cell line (HOSE) or human PBMCs. EV1 challenge was equally effective in the oncolysis of human ovarian cancer cells whether in monolayer or spheroidal environments. The therapeutic efficacy of EV1 was demonstrated by rapid reduction of tumor burden by a single viral intratumoral injection in SCID mice bearing multiple preformed s.c. xenografts. Using an in vivo i.p. human ovarian cancer xenograft model, administration of EV1 was further shown to significantly inhibit the formation and burden of ascites tumors. These findings demonstrate an important proof of principle for employing wild-type EV1 as a potential oncolytic agent in the control of human ovarian cancers.     

Knock-down of P-glycoprotein reverses taxol resistance in ovarian cancer multicellular spheroids

We previously established that multicellular ovarian cancer spheroids develop intrinsic multidrug resistance with the appearance of quiescent cell areas. p27 protein is a determinant of such resistance. However, the precise molecular basis of such resistance remains unknown. We demonstrated herein that these multicellular ovarian cancer spheroids expressed high levels of p27 and P-gp protein. Compared with monolayer cells, there is a significant increase in the resistance of spheroids cells to anticancer reagent Taxol. Antisense oligodeoxynucleotide not only mediated down-regulation of p27, but also P-gp expression in multicellular spheroids. Selective small interfering RNAs (siRNA) of P-gp with MDR1-targeted short hairpin RNAs (shRNA) expression vector sensitized the cells to Taxol. These results suggest that both p27 and P-gp can modulate Taxol sensitivity respectively, while p27 requires P-gp for its full function. Increased P-gp protein expression through p27 mediation is one of the major mechanisms of Taxol resistance in ovarian cancer multicellular spheroids.     

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.     

Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341.

Almost all known conventional cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than in killing tumor cells grown as monolayer cell cultures. This "multicellular resistance" reflects the relative intrinsic drug-resistant phenotype of most solid tumors growing in vivo and is due to factors such as limited drug penetration or reduced fractions of proliferating cells. Proteasome inhibitors such as PS-341, a dipeptide boronic acid analogue, represent an interesting new class of potential anticancer drugs, which are entering early-phase clinical trials. PS-341 has been found to have good broad-spectrum cytotoxic activity in the 60-monolayer cell line National Cancer Institute screen. However, because its relative potency has not been tested in spheroid systems, we analyzed the activity of PS-341 in a spheroid/solid tumor context using four different human ovarian carcinoma cell lines and three prostate carcinoma cell lines, respectively. We found, with one exception, that PS-341 showed equal or greater activity in spheroids than in the respective monolayer cell cultures, even in a prostate cancer spheroid model with a very low growth fraction. PS-341 induced apoptotic cell death in carcinoma cells in both culture systems. We also noted a decrease in XIAP protein, a member of the inhibitor of apoptosis (IAP) family of apoptosis inhibitors, and phosphorylation of Bcl-XL in PS-341-treated ovarian carcinoma cells. Furthermore, DNA fragmentation, a hallmark of apoptosis (in this case, induced by PS-341), was completely inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). Taken together, the results indicate that unlike most other known anticancer cytotoxic drugs, PS-341 appears to be as effective in killing tumor cells grown in the form of multicell spheroids as in killing tumor cells grown in monolayer cell culture. Hence, this compound has the potential to circumvent multicellular drug resistance and, as such, may show promising activity against solid tumors with low growth fractions in vivo, which are frequently intrinsically resistant to conventional cytotoxic anticancer drugs.     

Inhibition of phosphatidylinositol 3'-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models

Phosphatidylinositol 3'-kinase (PI3k) is implicated in a wide array of biological and pathophysiological responses. Thus, inhibiting molecules involved in its signal transduction pathway is a possible means of treating cancer. Our previous studies demonstrated that LY294002, a potent and selective PI3k inhibitor, decreases growth of ovarian carcinoma and ascites formation in an athymic mouse xenogeneic transplant model of ovarian cancer. However, the dose of LY294002 used to decrease tumor growth resulted in significant dermatological toxicity. We demonstrate herein that introduction of an active catalytic subunit of PI3k into an ovarian cancer cell line, and thus activation of the PI3k/AKT pathway, confers resistance to the effects of paclitaxel, one of the major drugs used in ovarian cancer therapy. The resistance to paclitaxel can be reversed in vitro by inhibition of PI3k. Therefore, we evaluated whether combined therapy with paclitaxel and LY294002 would result in increased efficacy and allow utilization of doses of LY294002 that do not induce dermatological toxicity. Two weeks after i.p. inoculation with OVCAR-3 ovarian cancer cells, mice were treated i.p. with LY294002 plus paclitaxel, each three times weekly on alternate days, for 4 weeks. Tumor burden in the LY294002 + paclitaxel, LY 294002 alone, and paclitaxel alone groups was reduced by 80% (P < 0.01), 38% (P < 0.05), and 51% (P < 0.05), respectively, compared with controls. Virtually no ascites developed in the combined treatment group; mean volume of ascites in the controls was 3.7 ml. Treatment with LY294002 alone reduced ascites by 70% (P < 0.01), whereas paclitaxel alone reduced ascites slightly but not significantly. No dermatological lesions or weight loss were observed in any treatment group. In vivo and in vitro morphological studies demonstrated that inhibition of PI3k enhanced paclitaxel-induced apoptosis in the human ovarian cancers. Our data suggest that a combination of a PI3k inhibitor and conventional chemotherapy may provide an effective approach to inhibiting tumor growth and ascites production in ovarian cancer with acceptable side effects.     

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.     

Resistance to chemotherapy mediated by TrkB in neuroblastomas

Neuroblastoma is a common childhood tumor derived from the peripheral nervous system. Favorable neuroblastomas usually express TrkA, the receptor for nerve growth factor (NGF), whereas unfavorable, MYCN-amplified neuroblastomas usually express TrkB and its ligand, brain-derived neurotrophic factor (BDNF). Here, we provide evidence that the TrkB-BDNF pathway is associated with enhanced survival and resistance to chemotherapy in neuroblastoma. We transfected the neuroblastoma line SH-SY5Y, which has endogenous expression of BDNF, with a full-length TrkB expression vector, and obtained clones with moderate or high levels of expression. Cells were exposed in vitro to chemotherapy agents used to treat neuroblastomas: doxorubicin, etoposide (VP16), and cisplatin. Chemoresistance was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell survival and by ELISA for cell death. In all cases, the TrkB-expressing subclones were more resistant to treatment than the parent line. Furthermore, when the TrkB tyrosine kinase was blocked with the Trk-specific inhibitor CEP-2563, or by neutralizing antibody to BDNF, sensitivity to chemotherapy was significantly increased. We also found constitutive phosphorylation of AKT at the Ser-473 site in TrkB transfectants, whereas there was only a minimal level of constitutive phosphorylation of AKT in SY5Y cells. These results show that the TrkB-BDNF pathway provides a survival advantage when exposed to DNA-damaging reagents, and, therefore, this autocrine pathway may play an important role in mediating the drug-resistant phenotype associated with TrkB-expressing neuroblastomas. Activation of PI3K/AKT survival pathway may contribute to the increased drug resistance in TrkB-expressing neuroblastomas.     

Aberrant methylation of breast and ovarian cancer susceptibility gene 1 in chemosensitive human ovarian cancer cells does not involve the phosphatidylinositol 3′‐kinase–Akt pathway

Methylation is an important silencing mechanism of breast and ovarian cancer susceptibility gene 1 (BRCA1) expression in sporadic ovarian cancer. However, the role of BRCA1 methylation in chemotherapy in sporadic ovarian cancer and the related pathways have not been understood completely. This study has determined the roles of BRCA1 hypermethylation in chemotherapy of sporadic ovarian cancer and its related signaling pathways. We used bisulfite sequencing, real‐time polymerase chain reaction, and western blotting to check the methylation state and expression levels of BRCA1 of the following cell lines: platinum‐sensitive human ovarian cancer cell line COC1, platinum‐resistant cell line COC1/DDP, SKOV‐3, and 5‐Aza‐dC treated COC1. The cisplatin sensitivity of ovarian cancer cells was examined by MTS (methyl‐thiazol tetrazolium) assay. Tumorigenicity in vivo and DDP‐based chemosensitivity were compared among the above cells. Phosphatidylinositol 3′‐kinase (PI3K)–Akt pathway activation in ovarian cancer cells was studied by western blotting. The frequency of BRCA1 methylation in the COC1 cell line was higher than in COC1/DDP and SKOV‐3 cell lines, whereas the mRNA and protein expression of BRCA1 were lower than in the COC1/DDP and SKOV‐3 cell lines. DNA demethylation decreased the chemosensitivity of COC1 cells and partially increased the expression levels of BRCA1. The activation of the PI3K‐Akt pathway was low in ovarian cancer cells. Our results indicate that hypermethylation of BRCA1 might play an important role in the chemosensitivity of ovarian cancer, and that the PI3K–Akt pathway is not involved in this response. (Cancer Sci 2010)     

Molecular description of a 3D in vitro model for the study of epithelial ovarian cancer (EOC)

Epithelial ovarian cancer (EOC) cell lines are useful tools for the molecular and biological characterization of ovarian cancer. The use of an in vitro multidimensional (3-D) culture model recapitulates some of the growth conditions encountered by tumor cells in vivo. Here we describe a molecular comparison of spheroid based 3D EOC models versus monolayer cultures and xenografts using cell lines from malignant ovarian tumors (TOV-21G and TOV-112D) and ascites (OV-90) previously established and characterized in our laboratory. Gene expression analyses of the three models were performed using the Affymetrix HG-U133A high density DNA array. Cluster analysis identified a set of genes that stratified expression profiles from the EOC cell lines grown as spheroids and xenografts from that of monolayer cultures. The gene expression analysis results were validated by Q-PCR analyses on an independent set of RNAs. Differential expression observed for the S100A6 gene between the monolayer, spheroid cultures and xenografts was confirmed at the protein level by immunohistochemistry. The analysis was extended to various ovarian tumor tissues using an EOC tissue array. This result represents an example of a gene that, if studied in vitro, is more representative of the in vivo disease in a 3D model rather than the monolayer culture. Identification of genes in spheroid models that mimic the in vivo tumor gene expression patterns may allow a better understanding of the community effect observed in human disease that is determined by direct or indirect interactions of cells with their environment or other surrounding cells.