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Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity.

ABSTRACT: OBJECTIVE:We propose that metastatic epithelial ovarian cancer (EOC) is a potential therapeutic target for the oncolytic agent, Myxoma virus (MYXV). METHODS:Primary EOC cells were isolated from patient ascites and cultured as adherent cells or in suspension using Ultra Low-Attachment dishes. MYXV expressing green fluorescent protein was used to infect cells and spheroids. Infection was monitored by fluorescence microscopy, viral titering and immunoblotting for M-T7 and M130 virus protein expression, and cell viability by alamarBlue assay. Akti-1/2 (5 ?M) and rapamycin (20 nM) were used to assay the role of PI3K-AKT signaling in mediating MYXV infection. RESULTS:Ascites-derived EOC cells grown in adherent culture are effectively killed by MYXV infection. EOC cells grown in suspension to form three-dimensional EOC spheroids readily permit MYXV entry into cells, yet are protected from the cytopathic effects of late MYXV infection. Upon reattachment (to model secondary metastasis), EOC spheroids are re-sensitized to MYXV-mediated oncolysis. The critical determinant that facilitates efficient MYXV infection is the presence of an activated PI3K-AKT signaling pathway. Treatment with the specific AKT inhibitor Akti-1/2 reduces infection of monolayer EOC cells and spheroids. Direct infection of freshly-collected ascites demonstrated that 54.5% of patient samples were sensitive to MYXV-mediated oncolytic cell killing. We also demonstrate that factor(s) present in ascites may negatively impact MYXV infection and oncolysis of EOC cells, which may be due to a down-regulation in endogenous AKT activity. CONCLUSIONS:Differential activity of AKT serves as the mechanistic basis for regulating MYXV-mediated oncolysis of EOC spheroids during key steps of the metastatic program. In addition, we provide the first evidence that MYXV oncolytic therapy may be efficacious for a significant proportion of ovarian cancer patients with metastatic disease.

SUBMITTER: Correa RJ

PROVIDER: S-EPMC4373451 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity.

Correa Rohann J M RJ Komar Monica M Tong Jessica G K JG Sivapragasam Milani M Rahman Masmudur M MM McFadden Grant G Dimattia Gabriel E GE Shepherd Trevor G TG

Gynecologic oncology 20120201 2

<h4>Objective</h4>We propose that metastatic epithelial ovarian cancer (EOC) is a potential therapeutic target for the oncolytic agent, Myxoma virus (MYXV).<h4>Methods</h4>Primary EOC cells were isolated from patient ascites and cultured as adherent cells or in suspension using Ultra Low-Attachment dishes. MYXV expressing green fluorescent protein was used to infect cells and spheroids. Infection was monitored by fluorescence microscopy, viral titering and immunoblotting for M-T7 and M130 virus ...[more]

PMID: 22306204

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Project description:Tumor cells not only show a vigorous metabolic state, but also reflect the disease progression and prognosis from their metabolites. To judge the progress and prognosis of ovarian cancer is generally based on the formation of ascites, or whether there is ascites recurrence during chemotherapy after ovarian cancer surgery. To explore the relationship between the production of ascites and ovarian cancer tissue, metabolomics was used to screen differential metabolites in this study. The significant markers leading to ascites formation and chemoresistance were screened by analyzing their correlation with the formation of ascites in ovarian cancer and the clinical indicators of patients, and then provided a theoretical basis. The results revealed that nine differential metabolites were screened out from 37 ovarian cancer tissues and their ascites, among which seven differential metabolites were screened from 22 self-paired samples. Sebacic acid and 20-COOH-leukotriene E4 were negatively correlated with the high expression of serum CA125. Carnosine was positively correlated with the high expression of serum uric acid. Hexadecanoic acid was negatively correlated with the high expression of serum γ-GGT and HBDH. 20a,22b-Dihydroxycholesterol was positively correlated with serum alkaline phosphatase and γ-GGT. In the chemotherapy-sensitive and chemotherapy-resistant ovarian cancer tissues, the differential metabolite dihydrothymine was significantly reduced in the chemotherapy-resistant group. In the ascites supernatant of the drug-resistant group, the differential metabolites, 1,25-dihydroxyvitamins D3-26, 23-lactonel and hexadecanoic acid were also significantly reduced. The results indicated that the nine differential metabolites could reflect the prognosis and the extent of liver and kidney damage in patients with ovarian cancer. Three differential metabolites with low expression in the drug-resistant group were proposed as new markers of chemotherapy efficacy in ovarian cancer patients with ascites.

Project description:Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n?=?13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and ?-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-?, ?A, ?B, anti-müllerian hormone, estrogen receptor [ER]-?, ER-?, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.

Project description:BackgroundThe tissue inhibitors of metalloproteinase (TIMPs) and their associated metalloproteinase (MMPs) are essential regulators of tissue homeostasis and are essential for cancer progression. This study analyzed the expression of TIMP-1,-2,-3 and the associated MMPs (MMP-2,-9,-11,-14) in different Stages, Grades and World Health Organization (WHO) classifications of serous ovarian tumors, ascites, ascites-derived cells from chemo-naïve (CN) and relapsed (CR) patients, and in ovarian cancer cell lines. The status of TIMPs and associated MMPs in response to chemotherapy treatment was assessed in cancer cell lines; TCGA data was interrogated to gauge TIMPs and associated MMPs as prognostic and platinum-response indicators.MethodsThe levels of TIMP-1, -2 and -3 were assessed by immunohistochemistry. The mRNA expression of TIMPs and MMPs was quantified by real time PCR (qRT-PCR). The chemosensitivity (IC50 values) to Cisplatin or Paclitaxel in cell lines was evaluated by MTT assay. The levels of TIMPs in ascites and cell lysates were analyzed by an ELISA assay.ResultsThe expression of TIMP-2 was significantly upregulated in Type 2 compared to Type 1 tumors and normal/benign ovarian tissues. TIMP-3 expression was significantly enhanced in Stage III, Grade 3 and Type 2 tumors compared to normal/benign ovarian tissues. The mRNA expression of MMP-9,-11 and -14 was significantly upregulated in Stage IV compared to normal/benign ovarian tissues. The expression of TIMP-1 was highest, followed by TIMP-2 and then TIMP-3 in CN ascites. At the cellular level, TIMP-2 mRNA expression was significantly higher in CN compared to CR epithelial cells in patients. The expression of TIMP-1 and -2, MMPs and cancer stem cells (CSCs) were upregulated in response to chemotherapy treatments in cancer cell lines. Interrogation of the TCGA dataset suggests shifts in platinum responses in patients consistent with genetic alterations in TIMP-2, -3 and MMP-2, -11 genes in tumors; and decreased overall survival (OS) and progression-free survival (PFS) in patients with altered MMP-14 genes.ConclusionsTIMPs and related MMPs are differentially expressed in serous ovarian tumors, ascites, ascites-derived cells and ovarian cancer cell lines. Chemotherapy treatment modulates expression of TIMPs and MMPs in association with increased expression of genes related to cancer stem cells.

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Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity.

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Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity.

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