Monoclonal antibody uptake in B-cell lymphomas: Experimental studies in nude mouse xenografts

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.     

Radioimmunotherapy of small-cell lung cancer xenografts using131I-labelled anti-NCAM monoclonal antibody 123C3

We have studied the therapeutic efficacy of¹³¹I-labelled monoclonal antibody 123C3 in human small-cell lung carcinoma xenografts established from the NCI-H69 cell line in nude mice. Several radiation does were administered intraperitoneally and different treatment schedules were tested. The maximal tolerated dose, 2×500 Ci, resulted in complete remission of tumours smaller than 200 mm³ and long-lasting remission (more than 135 days) of the larger tumours. In control experiments, treatment with unlabelled monoclonal antibody 123C3 did not affect the tumour growth rate, while the effect of radiolabelled non-relevant, isotype-matched, monoclonal antibody M6/1 was minor and transient. Regrowth of the tumours occurred in all cases and could not be attributed to loss of neural cell adhesion molecule (NCAM) expression. Tumour recurrence is probably caused by insufficient radiation dosage. Radiation-induced toxicity was monitored by assessment of weight and bone marrow examination. Weight loss was observed in all treatment groups, but the mice regained their initial weight within 14 days, except for the group receiving the highest radiation dose (3×600 Ci). In this group all mice died as a result of radiotoxicity. Of the mice injected with 600 Ci radiolabelled control antibody, 50% died within 2 weeks after administration. Apparently the higher uptake of the radiolabelled monoclonal antibody in the tumour reduced systemic radiation toxicity.     

Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01

mAb hCTM01 binds a carcinoma-associated antigen, the MUC1 gene product. The antigen is also present in the circulation, and administration of ¹¹¹In-labelled hCTM01 results in the formation of immune complexes with enhanced accumulation in the liver. To avoid the unwanted effect of circulating radioactive immune complexes, a strategy to remove the circulating antigen was investigated using a split-dosage schedule. Eleven patients suspected of having ovarian carcinoma were injected with 1 mg/kg unlabelled hCTM01, 1 h before receiving 0.1 mg/kg ¹¹¹In-labelled hCTM01 (100 MBq). The amount of radioactivity was determined in resected tumour tissue, various normal tissues and blood samples obtained at laparotomy 6 days postinjection (p.i.). In all patients, the circulating antigen decreased to its nadir after the unlabelled antibody infusion and immune complex formation was demonstrated. Uptake in tumour deposits 6 days p.i. was 11.1 times higher than in normal tissues (P<0.0001) and 5.9 times higher than in blood (P<0.0001). ¹¹¹In activity in liver tissue was comparable to ¹¹¹In uptake in tumour tissue, and considerably lower than previously reported in patients not pretreated with unlabelled antibody. The split-dosing strategy would appear to be advantageous for use of hCTM01 as a specific carrier for the delivery of cytotoxic agents to patients with ovarian cancer. Received: 12 February 1998 / Accepted: 30 April 1998     

长链非编码RNA-H19 在妇科恶性肿瘤细胞的糖代谢中的作用

目的:检测长链非编码RNA-H19在常见的妇科恶性肿瘤中的表达,并探讨其在肿瘤细胞糖代谢过程中发挥的可能作用。方法:以ARK2子宫内膜癌细胞株和OVCAR-3上皮性卵巢癌细胞株作为实验对象;利用脂质体转染技术,将特异性靶向小干扰RNA序列转染进细胞;利用TRIzol法提取样品的总RNA,并通过逆转录和实时定量PCR技术检测样品中目的基因的表达;利用荧光标记的非放射性2-NBDG评估肿瘤细胞的葡萄糖摄取能力;采用方差齐性检验和t检验进行统计学分析。结果:H19在ARK2和OVCAR-3细胞中的表达均较高。在ARK2和OVCAR-3细胞中,si H19抑制H19表达的效率分别为34.02%和30.30%。是否添加Insulin对于转染si Con的肿瘤细胞的糖摄取能力无明显影响,但是si H19转染后的ARK2和OVCAR-3细胞,在经Insulin处理后糖摄取能力均明显增强。结论:ARK2和OVCAR-3这两种细胞是研究H19在妇科恶性肿瘤中机制的较为理想的实验对象,ARK2和OVCAR-3细胞本身存在胰岛素抵抗,外源性抑制H19表达后,细胞的胰岛素抵抗有所改善,H19或成为我们改善妇科恶性肿瘤患者机体葡萄糖代谢的标记物。     

Immunoscintigraphic localization of renal tumours in an extracorporeal perfusion model with a monoclonal antibody against γ-glutamyltransferase

Summary Monoclonal antibody 138H11 against human -glutamyltransferase has been shown to react immunohistochemically with 98% of all tested clear-cell type and chromophilic renal cell carcinomas, but not with renal chromophobic carcinomas, Duct-Bellini carcinomas or oncocytomas. In normal kidney the target epitopes of mAb 138H11 are located in the luminal brush-border membrane of proximal tubule cells, whereas in renal carcinomas the epitopes are found surrounding the whole tumour cells. These results form the basis of the present immunoscintigraphic study designed to evaluate mAb 138H11 in an extracorporeal perfusion model. Immediately after nephrectomy, human tumour-bearing kidneys were perfused with^99mTc-labelled mAb 138H11 in Euro-Collins solution. High specific uptake in 4/4 renal clear cell carcinomas could be demonstrated by planar immunoscintigraphy and single-photon-emission computed tomography, regions of interest investigation and immunohistochemistry. In contrast, a perfused oncocytoma showed up as an unlabelled lesion. The results indicate a possible use for mAb 138H11 in immunoscintigraphy or even therapy, provided high tumour uptake can be confirmed in patients.     

Bispecific-monoclonal-antibody-directed lysis of ovarian carcinoma cells by activated human T lymphocytes

Summary Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3KS1/4 and OKT3KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h⁵¹Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10–10000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.     

Imaging of human ovarian tumour xenografts in nude mice using a novel 111In-labelled monoclonal antibody (10B)

Monoclonal antibody (mAb) 10B, directed against the human ovarian adenocarcinoma cell line, HEY, was conjugated with cyclic DTPA anhydride and labelled with ¹¹¹In. The biodistribution of ¹¹¹In-DTPA-10B was determined in non-tumour bearing mice and mice bearing subcutaneous (s.c.) and intraperitoneal (i.p.) HEY tumours. The radiolabel was preferentially targeted to s.c. and i.p. tumours in comparison with a control mAb, ¹¹¹In-DTPA-2G3, which does not bind to HEY cells. Among normal organs, the predominant uptake of radiolabel was into liver and kidney. Subcutaneous tumours were successfully imaged using external gamma scintigraphy following i.v. injection of ¹¹¹In-DTPA-10B. The results suggest that ¹¹¹In-DTPA-10B may be a useful agent for the diagnostic imaging of tumour masses in patients with ovarian cancer.     

Interleukin-10 in serous ovarian carcinoma cell lines

 Interleukin-10, one of the most potent anti-inflammatory cytokines, is expressed in ovarian carcinomas in vivo. In contrast to the high levels of IL-10 in ascites and tumour tissue, the expression of this cytokine appears to be a rare event in ovarian carcinoma cell lines in vitro. Virtually nothing is known about the regulation of IL-10 expression in ovarian carcinoma cell lines. We investigated the expression of IL-10 in four cell lines originally derived from ovarian serous adenocarcinoma: OVCAR-3, SKOV-3, CAOV-3 and OAW-42. IL-10-specific mRNA was detected in OVCAR-3 and only this cell line produced IL-10 constitutively under serum-free conditions as well as in serum-containing medium. Our studies on the regulation of IL-10 secretion in OVCAR-3 revealed that (1) proinflammatory stimuli IL-1β and TNF-α, but not LPS, enhance IL-10 secretion, (2) IL-6 has no influence on the release of IL-10, (3) prostaglandin E2 influences neither the spontaneous nor the TNF-α- or IL-1β-stimulated IL-10 production and (4) interferon-γ inhibits IL-10 secretion. We conclude that only a minority of serous ovarian carcinoma cells maintain the ability to produce IL-10 in vitro. Our data on the regulation of IL-10 production in OVCAR-3 indicate that ovarian carcinoma cells share some, but not all, of the regulatory features typical for the monocytic IL-10 secretion. Received: 1 February 2001 / Accepted: 29 March 2001     

High expression of integrin αvβ3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvβ3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvβ3 receptors ([9.8?±?2.5]?×?10⁴/cell) compared to the well-characterized cell line U87-MG ([5.2?±?1.4]?×?10⁴/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvβ3 receptor density is actually lower (11?±?3 versus 18?±?6?receptors/µm²). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (～680?nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2?±?0.4 higher than the monovalent probe, which in turn was 2.2?±?0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvβ3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.     

Antibody-dependent difference in biodistribution of monoclonal antibodies in animal models and humans

 The study was designed to clarify the difference in pharmacokinetics of monoclonal antibodies (mAb) in animal models and humans, and to elucidate the applicability of animal models. ^99mTc-labeled murine mAb – against carcinoembryonic antigen (designated BW431/26), and neural cell adhesion molecule (NE150) – and one chimeric mouse/human mAb against nonspecific cross-reacting antigen (chNCA) were administered i.v. to normal mice and athymic mice (370 kBq, 400 ng) xenografted with human cancer cells expressing antigens, and into patients with tumor (925 MBq, 1 mg). The biodistribution of two of the three mAb (not ^99mTc-BW431/26) differed clearly in mice and patients. ^99mTc-NE150 showed specific uptake in xenografted tumor and otherwise a normal biodistribution; however, clinical examination showed increased uptake in the liver with rapid blood clearance (mean α half-life = 31.1 min) compared with ^99mTc-BW431/26 (28.4 h). ^99mTc-chNCA demonstrated increased blood clearance and renal excretion in both normal and athymic mice, with accumulation in tumors. Clinical examination showed rapid blood clearance (mean α half-life = 6.4 min) and increased uptake in the liver. High-performance liquid chromatographic analysis of ^99mTc-chNCA revealed the immune complex in blood, suggesting uptake of the complex by the reticuloendothelial cells. The biodistribution of radiolabeled mAb in animal and human models was variable and specific for each of the three mAb. The results of animal studies with mAb should be evaluated carefully before being extrapolated to humans, on the basis of the nature of the mAb and interacting substances. Received: 9 April 1997 / Accepted 3 March 1998     

片仔癀对人卵巢癌OVCAR-3细胞增殖、凋亡及周期的影响

目的：研究片仔癀对人卵巢癌细胞株OVCAR-3增殖抑制作用,及其对细胞周期、细胞凋亡的影响。方法：采用MTT法观察片仔癀对人卵巢癌细胞株OVCAR-3细胞的增殖抑制率,流式细胞仪检测细胞凋亡及细胞周期,westem—blot检测相关蛋白的表达。结果：片仔癀以剂量依赖式抑制人卵巢癌细胞株OVCAR-3细胞增殖,片仔癀250、500、1000μg·mL-1作用于OVCAR-3细胞24h后,其早期凋亡率分别为6．6％、30．9％、43．2％,而对照组为0％,其诱导凋亡作用呈现剂量依赖性;细胞积聚在G0／G1期,同时S期细胞比例减少;Akt、PARP、CDK6表达下调。结论：片仔癀可以抑制OVCAR-3细胞增殖及诱导细胞凋亡作用,并能阻滞细胞于G0／G1期,有望成为卵巢癌治疗药。     

An Antimitotic and Antivascular Agent BPR0L075 Overcomes Multidrug Resistance and Induces Mitotic Catastrophe in Paclitaxel-Resistant Ovarian Cancer Cells

Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC₅₀ = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.     

Protectors against doxorubicin-induced cardiotoxicity: Flavonoids

Doxorubicin is a widely used anthracycline anticancer agent. Its use may cause cardiomyopathy: in fact, the development of cumulative dose-related cardiotoxicity forms the major limitation of clinical doxorubicin use. We therefore searched for protective agents that combine iron-chelating and oxygen radical-scavenging properties. Moreover, any novel protector should not interfere with the cytostatic activity of doxorubicin. After extensive in vitro screening we found that flavonoids could serve this purpose. In particular 7-monohydroxyethylrutoside almost completely protected against the negative inotropic action of doxorubicin in the electrically paced mouse left atrium model. In vivo it gave full protection at 500 mg/kg intraperitoneally against the doxorubicin-induced ST-interval lengthening in the ECG. Moreover, this protector did not influence the antitumor effect of doxorubicin either in vitro using the human ovarian cell lines A2780 and OVCAR-3 and the human breast cancer cell line MCF-7 or in vivo in A2780 and OVCAR-3 subcutaneous xenografts in nude mice. Comparison of various iron chelators suggest that iron, in contrast to the general assumption, might not play a crucial role in the oxidative stress-induced toxicity of doxorubicin. Moreover, incubation of vascular endothelial cells with doxorubicin produced overexpression of adhesion molecules, which could be inhibited by 7-monohydroxyethylrutoside. From a study in human volunteers, we conclude that an intravenous dose of 1500 mg/m² of 7-monohydroxyethylrutoside is feasible and is safe to be investigated as protection against doxorubicin-induced cardiotoxicity.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Carbohydrate vaccines that induce antibodies against cancer. 1. Rationale

  Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant form of Pseudomonas exotoxin A lacking the cell-binding domain (ETA_{252 – 613}) was prepared, and its effect on lung cancer cell lines examined. MOC31-ETA_{252 – 613} was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the numberof clonogenic tumour cells from cultures by factors of 10⁴ and 10⁵ during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm³) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm³). This finding indicates that MOC31-ETA_{252 – 613} may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease. Received: 31 October 1996 / Accepted: 5 December 1996     

LPA-induced migration of ovarian cancer cells requires activation of ERM proteins via LPA1 and LPA2

Lysophosphatidic acid (LPA) has been implicated in the pathology of human ovarian cancer. This phospholipid elicits a wide range of cancer cell responses, such as proliferation, trans-differentiation, migration, and invasion, via various G-protein-coupled LPA receptors (LPARs). Here, we explored the cellular signaling pathway via which LPA induces migration of ovarian cancer cells. LPA induced robust phosphorylation of ezrin/radixin/moesin (ERM) proteins, which are membrane-cytoskeleton linkers, in the ovarian cancer cell line OVCAR-3. Among the LPAR subtypes expressed in these cells, LPA₁ and LPA₂, but not LPA₃, induced phosphorylation of ERM proteins at their C-termini. This phosphorylation was dependent on the Gα_12/13/RhoA pathway, but not on the Gα_q/Ca²⁺/PKC or Gα_s/adenylate cyclase/PKA pathway. The activated ERM proteins mediated cytoskeletal reorganization and formation of membrane protrusions in OVCAR-3 cells. Importantly, LPA-induced migration of OVCAR-3 cells was completely abolished not only by gene silencing of LPA₁ or LPA₂, but also by overexpression of a dominant negative ezrin mutant (ezrin-T567A). Taken together, this study demonstrates that the LPA₁/LPA₂/ERM pathway mediates LPA-induced migration of ovarian cancer cells. These findings may provide a potential therapeutic target to prevent metastatic progression of ovarian cancer.     

Expression and roles of Slit/Robo in human ovarian cancer

The Slit glycoproteins and their Roundabout (Robo) receptors regulate migration and growth of many types of cells including human cancer cells. However, little is known about the expression and roles of Slit/Robo in human ovarian cancer. Herein, we examined the expression of Slit/Robo in human normal and malignant ovarian tissues and its potential participation in regulating migration and proliferation of human ovarian cancer cells using two ovarian cancer cell lines, OVCAR-3 and SKOV-3. We demonstrated that Slit2/3 and Robo1 were immunolocalized primarily in stromal cells in human normal ovaries and in cancer cells in many histotypes of ovarian cancer tissues. Protein expression of Slit2/3 and Robo1/4 was also identified in OVCAR-3 and SKOV-3 cells. However, recombinant human Slit2 did not significantly affect SKOV-3 cell migration, and OVCAR-3 and SKOV-3 cell proliferation. Slit2 also did not induce ERK1/2 and AKT1 phosphorylation in OVCAR-3 and SKOV-3 cells. The current findings indicate that three major members (Slit2/3 and Robo1) of Slit/Robo family are widely expressed in the human normal and malignant ovarian tissues and in OVCAR-3 and SKOV-3 cells. However, Slit/Robo signaling may not play an important role in regulating human ovarian cancer cell proliferation and migration.     

Experimental therapy of ovarian cancer with synthetic makaluvamine analog: in vitro and in vivo anticancer activity and molecular mechanisms of action

The present study was designed to determine the biological effects of novel marine alkaloid analog 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) on human ovarian cancer cells for its anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. Human ovarian cancer cells (A2780 and OVCAR-3), and Immortalized non-tumorigenic human Ovarian Surface Epithelial cells (IOSE-144), were exposed to FBA-TPQ for initial cytotoxicity evaluation (via MTS assay kit, Promega). The detailed in-vitro (cell level) and in-vivo (animal model) studies on the antitumor effects and possible underlying mechanisms of action of the compounds were then performed. FBA-TPQ exerted potent cytotoxicity against human ovarian cancer A2780 and OVCAR-3 cells as an effective inhibitor of cell growth and proliferation, while exerting lesser effects on non-tumorigenic IOSE-144 cells. Further study in the more sensitive OVCAR-3 cell line showed that it could potently induce cell apoptosis (Annexin V-FITC assay), G2/M cell cycle arrest (PI staining analysis) and also dose-dependently inhibit OVCAR-3 xenograft tumors' growth on female athymic nude mice (BALB/c, nu/nu). Mechanistic studies (both in vitro and in vivo) revealed that FBA-TPQ might exert its activity through Reactive Oxygen Species (ROS)-associated activation of the death receptor, p53-MDM2, and PI3K-Akt pathways in OVCAR-3 cells, which is in accordance with in vitro microarray (Human genome microarrays, Agilent) data analysis (GEO accession number: GSE25317). In conclusion, FBA-TPQ exhibits significant anticancer activity against ovarian cancer cells, with minimal toxicity to non-tumorigenic human IOSE-144 cells, indicating that it may be a potential therapeutic agent for ovarian cancer.     

Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition.     

Combined application of camptothecin and the guanylate cyclase activator YC-1: Impact on cell death and apoptosis-related proteins in ovarian carcinoma cell lines

Camptothecin analogs and guanylate cyclase activator YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] have been shown to induce apoptosis in cancer cells. However, the combined effect of camptothecin analogs and YC-1 on the viability of epithelial ovarian cancer cells remains uncertain. We assessed the combined effect of YC-1 on the camptothecin toxicity in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Camptothecin and YC-1 induced apoptosis in OVCAR-3 and SK-OV-3 cells in a dose- and time-dependent manner. Both compounds induced nuclear damage, decreased Bid and Bcl-2 protein levels, enhanced cytochrome c release, activated caspase-3 and upregulated tumor suppressor p53. Camptothecin decreased Bax protein levels, whereas YC-1 increased Bax levels. YC-1 enhanced the camptothecin-induced changes in the apoptotic protein levels and increased apoptotic effect of camptothecin on ovarian carcinoma cell lines. The results suggested that YC-1 may enhance a camptothecin toxicity against ovarian carcinoma cell lines by increasing activation of the caspase-8 and Bid pathway as well as activation of the mitochondria-mediated apoptotic pathway, leading to cytochrome c release and subsequent caspase-3 activation. Combination of camptothecin analogs and YC-1 may provide a therapeutic benefit against ovarian adenocarcinoma.