Ovarian cancer linked to Lynch syndrome typically presents as early-onset, non-serous epithelial tumors

Objective

Heredity is a major cause of ovarian cancer and during recent years the contribution from germline mismatch repair (MMR) gene mutations linked to Lynch syndrome has gradually been recognized.

Methods

We characterized clinical features, tumor morphology and mismatch repair defects in all ovarian cancers identified in Swedish and Danish Lynch syndrome families.

Results

In total, 63 epithelial ovarian cancers developed at mean 48 (range 30-79) years of age with 47% being early stage (FIGO stage I). Histologically, endometrioid (35%) and clear cell (17%) tumors were overrepresented. The underlying MMR gene mutations in these families affected MSH2 in 49%, MSH6 in 33% and MLH1 in 17%. Immunohistochemical loss of the corresponding MMR protein was demonstrated in 33/36 (92%) tumors analyzed.

Conclusion

The combined data from our cohorts demonstrate that ovarian cancer associated with Lynch syndrome typically presents at young age as early-stage, non-serous tumors, which implicates that a family history of colorectal and endometrial cancer should be specifically considered in such cases.     

Differential in vitro sensitivity to patupilone versus paclitaxel in uterine and ovarian carcinosarcoma cell lines is linked to tubulin-beta-III expression

Objective

To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine and ovarian carcinosarcomas (CS).

Methods

Five primary carcinosarcoma cell lines, two from uterine and three of ovarian origin, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. To identify potential mechanisms underlying the differential sensitivity/resistance to patupilone, expression levels of β-tubulin III (TUBB3) were determined with quantitative-real-time-polymerase-chain-reaction (q-RT-PCR) in primary uterine and ovarian CS cell lines and in 26 uterine and 9 ovarian CS fresh-frozen-tissues.

Results

No appreciable difference in sensitivity to patupilone versus paclitaxel was noted in ovarian CS cell lines, or when uterine and ovarian CS cell lines were compared in their response to paclitaxel. In contrast, uterine CS cell lines were found to be significantly more sensitive to patupilone than to paclitaxel (P < 0.002) and demostrated lower IC_50s to patupilone (range 0.76-0.93 nM) when compared to ovarian CS (range 1.9-3.4 nM, p < 0.05). Higher levels of TUBB3 were detected in uterine CS cell lines and fresh frozen tissues when compared to ovarian CS (P < 0.05).

Conclusions

Uterine CS cell lines are significantly more sensitive than ovarian CS cell lines to patupilone versus paclitaxel. High expression of TUBB3 is associated with sensitivity to patupilone in primary CS cell lines and may act as a genetic marker to predict chemotherapy efficacy. Patupilone may represent a promising drug in the treatment of this subset of rare but highly aggressive gynecological tumors.     

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination

Objectives

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines.

Methods

Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed.

Results

Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively.

Conclusions

Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.     

Novel nanotechnology approaches to diagnosis and therapy of ovarian cancer

Objective

Ovarian carcinoma is the leading cause of death from gynecologic malignancies, which is a direct outcome of missing its diagnosis at an early stage. Approximately 75% of ovarian cancer patients are initially diagnosed with disseminated intra-abdominal disease (stages III-IV) when ~ 30% of patients have a 5-year survival rate. In addition to the challenge of early detection of ovarian cancer, its therapy presents several challenges including the route of therapy, resistance to therapy with recurrence of cancer, and specific targeting of ovarian cancer to reduce cytotoxic side effects.

Methods

We reviewed recent literature employing nanotechnology approaches to diagnosis and therapy of ovarian cancer.

Results

Recent innovations in nanotechnology with applications in cancer diagnostics and therapy help circumvent many pre-existing problems with conventional chemotherapy and present new ways of diagnosis and therapy.

Conclusions

Nanotechnology has promising potential in enhancing early detection of ovarian cancer and treatment of recurrent disease.     

Epigenetic silencing of miR-130b in ovarian cancer promotes the development of multidrug resistance by targeting colony-stimulating factor 1

Objective

The purpose of this study was to investigate the role of miR-130b in the development of multidrug-resistant ovarian cancer.

Methods

The expression of miR-130b was assessed in ovarian tissues and cell lines by qRT-PCR. In vitro, miR-130b level was manipulated by transfection with mimics or inhibitors. Methylation level of miR-130b was evaluated by quantitative methylation-specific PCR (qMSP). CSF-1 expression in ovarian tissues and cells was determined by qRT-PCR, immunohistochemistry and ELISA, respectively. CSF-1 regulated by miR-130b was detected using Dual Luciferase Reporter system.

Results

Down-regulation of miR-130b in ovarian cancer was associated with FIGO III-IV clinical stages and poorer histological differentiation. MiR-130b was downregulated in multidrug resistant ovarian cancer cells. Restoration of miR-130b expression could sensitize these cells to anticancer drugs. MiR-130b hypermethylation was found in ovarian cancer tissues as well as in drug resistant cell lines and the methylation level was negatively correlated with its expression. Demethylation with 5-aza-CdR led to reactivation of miR-130b expression in drug resistant ovarian cancer cell lines concomitant with increase of sensibility to cisplatin and taxol. CSF-1 expression was negatively associated with miR-130b level in ovarian tissues and cell lines. Luciferase assay validated CSF-1 is a direct target of miR-130b. Knock-down of CSF-1 sensitized ovarian cancer cells to anticancer drugs and could partially attenuate the resistance inducing effect of miR-130b inhibitors.

Conclusions

Downregulation of miR-130b promotes the development of multidrug resistant ovarian cancer partially by targeting the 3′-UTR of CSF-1, and the silencing of miR-130b may be mediated by DNA methylation.     

High-mobility group protein B1 (HMGB1) is a novel biomarker for human ovarian cancer

Background

High mobility group box l (HMGB1), a nuclear and extracellular protein, is implicated in some physiologic and pathologic conditions. In this study, we investigated the expression and function of HMGB1 in ovarian cancer.

Methods

cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate HMGB1 expression in a total of 100 ovarian tissue specimens. In functional assays, effects of HMGB1 knockdown on the biological behavior of ovarian cancer cells were investigated.

Results

HMGB1 was overexpressed in the highly invasive subclone compared with the low invasive subclone. High HMGB1 expression was associated with poor clinicopathologic features. Knockdown of HMGB1 expression significantly suppressed ovarian cancer cell proliferation accompanied by decreased cyclin D1 and PCNA expression, and inhibited cell migration and invasion accompanied by decreased MMP2 and MMP9 activities.

Conclusion

HMGB1 is a newly identified gene overexpressed in ovarian cancer and associated with poor clinicopathologic features. HMGB1 may serve as a new biomarker and a therapeutic target for ovarian cancer in the future.     

Dasatinib (BMS-35482) has synergistic activity with paclitaxel and carboplatin in ovarian cancer cells

Purpose

To explore the activity of dasatinib alone and in combination with paclitaxel and carboplatin in ovarian cancer cells and to determine if dasatinib activity can be predicted based on evaluation of the SRC pathway.

Experimental design

Microarray analysis was performed for IGROV1, OVCAR3, A2780 and SKOV3 ovarian cancer cells and the status of the genomic SRC signature pathway was determined. Cells were treated with carboplatin, paclitaxel and dasatinib individually and in combination. Pre- and post-treatment phospho-SRC (pSRC) and SRC protein expression was determined. Dose-response curves were constructed, and drug interaction was assessed by the Combination Index (CI) method.

Results

SRC protein expression levels reflected the SRC pathway genomic signature in the cell lines with the lowest (SKOV3) and highest (IGROV1) pathway expression, but not in those with intermediate expression (OVCAR3, A2780). Dasatinib treatment caused loss of pSRC in all cell lines, with 50% growth inhibition for IGROV1 at 70 nM, OVCAR3 at 34 nM, A2780 at 4.1 μM and SKOV3 at 530 nM. Dasatinib combined with cytotoxics yielded a synergistic effect (CI = 0.46 to 0.79) in all cell lines except SKOV3.

Conclusion

Dasatinib in combination with standard chemotherapeutic agents appears to interact in a synergistic manner in some ovarian cancer cell lines. Further research is needed to evaluate tumor cell characteristics which predict response to dasatinib.     

Inhibition of cell growth and induction of apoptosis in ovarian carcinoma cell lines CaOV3 and SKOV3 by natural withanolide Withaferin A

Objective

Withaferin A, a natural withanolide, has shown anti-cancer properties in various cancers including breast cancer, but its effects in ovarian cancer remain unexplored. Notch 1 and Notch3 are critically involved in ovarian cancer progression. We decided to examine the effects of Withaferin A in ovarian carcinoma cell lines and its molecular mechanism of action including its regulation of Notch.

Methods

The effects of Withaferin A were examined in CaOV3 and SKOV3 ovarian carcinoma cell lines using MTS assay, clonogenic assay, annexin V/propidium iodide flow cytometry, and cell cycle analysis. Western analysis was conducted to examine the molecular mechanisms of action.

Results

Withaferin A inhibited the growth and colony formation of CaOV3 and SKOV3 cells by inducing apoptosis and cell cycle arrest. These changes correlated with down-regulation of Notch1, Notch3, cdc25C, total and phosphorylated Akt, and bcl-2 proteins.

Conclusions

Withaferin A inhibits CaOV3 and SKOV3 ovarian carcinoma cell growth, at least in part by targeting Notch1 and Notch3.     

Metformin targets ovarian cancer stem cells in vitro and in vivo

Purpose

Studies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC.

Methods

The impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts.

Results

Metformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis.

Conclusions

Metformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.     

Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin

Objectives

In recent years, cancer stem cells (CSCs) have been reported to be correlated with chemoresistance and may also be enriched in side populations (SPs). In this study, the relationship between resistance to paclitaxel (PTX) and cisplatin (CDDP) and side populations was examined in three parental PTX- and CDDP-sensitive ovarian cancer cell lines (2008, KF28, and TU-OM-1) and several other cell lines derived from these as well as the additional effects of interferon-alpha (INF-α).

Methods

SP of three different parental cell lines and PTX- and/or CDDP-resistant cell lines derived from these was analyzed with flow cytometry. The expression of ABCB1 and ABCG2 in KF28 and its derived cell lines was examined. Additional cell-death effect of INF-α with PTX was also examined.

Results

In the three parental cell lines and the PTX-sensitive cell lines derived from these lines, SP was very low. Conversely, in PTX-resistant cell lines, regardless of CDDP resistance, SP increased. ABCB1 was strongly expressed in the PTX-resistant cells, but not in their parental lines, which are sensitive to PTX. While INF-α showed only slight enhancement of the cell-death effect of PTX in PTX-sensitive cells, INF-α itself strongly induced apoptosis in PTX-resistant cells regardless of PTX concentration.

Conclusions

The SP could be correlated with resistance to PTX. SP could be a target of INF-α, and resistance to PTX might be overcome by INF-α.     

Green tea for ovarian cancer prevention and treatment: a systematic review of the in vitro, in vivo and epidemiological studies

Objective

This systematic review was conducted to examine the effects of green tea or green tea components on the prevention and progression of epithelial ovarian cancer.

Methods

Using Medline, EMBASE and SciVerse (last researched: July 2011), we retrieved 22 articles including 5 epidemiological studies.

Results

In epithelial ovarian cancer cell lines, green tea and green tea components have been shown to downregulate the expression of proteins involved in inflammation, cell signalization, cell motility and angiogenesis. Green tea and green tea components would induce apoptosis and could potentiate the effects of cisplatin, a chemotherapeutic agent. In human observational studies, significant associations between green tea intake and both decreased ovarian cancer occurrence and better prognosis were reported.

Conclusions

Available literature suggests potential molecular targets for green tea in ovarian cancer treatment and also provides data supporting the clinical evaluation of the role of green tea or green tea components in ovarian cancer prevention and treatment.     

Inhibin/activin betaE-subunit in uterine endometrioid adenocarcinoma and endometrial cancer cell lines: from immunohistochemistry to clinical testing?

Objective

Inhibins and activins are important regulators of the female reproductive system and have also been described to be involved in gynecologic cancer development. A new inhibin/activin subunit betaE has been identified, but only limited data on its expression in human uterine adenocarcinomas and endometrial cancer cell lines exist.

Methods

A series of 223 uterine endometrial adenocarcinomas were immunohistochemically analyzed with a specific antibody against the inhibin betaE-subunit. In addition, established endometrial cancer cell lines were analyzed for the synthesis of the inhibin betaE subunit by immunofluorescence and RT-PCR analysis.

Results

In this analysis, the inhibin betaE staining intensity was associated with histological grading along with a significant decrease in cases with ovarian invasion. However, inhibin-betaE did not affect patient survival nor did it constitute an independent prognostic parameter in endometrial adenocarcinoma patients. Additionally, the inhibin/activin betaE-subunit was found to be expressed in the human endometrial carcinoma cell lines HEC1a, HEC1b, Ishikawa, and RL95-2, although the level of expression was found to be highly differing among the cancer cell lines tested.

Conclusion

The isolated analysis of the inhibin betaE subunit might be of minor prognostic value in identifying high-risk patients. However, its differential expression in cancer of different histological differentiation and ovarian metastasis suggests a putative but yet undefined role in endometrial carcinogenesis. Whether this novel and not well studied inhibin subunit has a substantial function in the pathogenesis and malignant transformation of human endometrium is still under investigation.     

Luteinizing hormone upregulates survivin and inhibits apoptosis in ovarian epithelial tumors

Objective

Luteinizing hormone (LH) plays an important role in the development of ovarian cancer, and has been shown to inhibit apoptosis in ovarian cancer cells. Similarly, survivin is a molecule that has been shown to inhibit apoptosis in other types of cancer. Therefore, the aim of this study was to determine whether survivin can be induced by LH in ovarian cancer, and whether this induction influences the sensitivity of ovarian cancers to chemotherapy.

Study design

Survivin expression was monitored using western blot assays, and flow cytometry was used to detect the effects of cisplatin on the induction of apoptosis by LH. MTT assays were also used to analyze rates of cell proliferation.

Results

Administration of LH in vitro induced survivin expression in a dose-dependent manner. Moreover, this signaling was dependent on the ERK1/2 signaling pathway. LH also blocked apoptosis induced by cisplatin.

Conclusion

These results suggest that LH influences the sensitivity of ovarian cancer cells to chemotherapy via signaling to inhibit apoptosis that also upregulates survivin.     

Modulation of redox signaling promotes apoptosis in epithelial ovarian cancer cells

Objective

Epithelial ovarian cancer (EOC) cells are known to be resistant to apoptosis through a mechanism that may involve alteration in their redox balance. NADPH oxidase is a major source of intracellular superoxide, which is converted to the less toxic product by superoxide dismutase (SOD). Superoxide contributes to hypoxia inducible factor (HIF)-1α stabilization. We sought to determine the effects of inhibiting the generation of intracellular reactive oxygen species (ROS) on apoptosis of EOC cells.

Methods

Diphenyleneiodonium (DPI), an irreversible ROS inhibitor, was used to inhibit the generation of ROS in EOC cell lines, SKOV-3 and MDAH-2774, followed by assessment of apoptosis, NADPH oxidase, SOD3 and HIF-1α expression. A combination of immunohistochemistry, immunoprecipitation/western blot, and real-time RT-PCR were utilized to evaluate the expression of these enzymes in EOC cells as well as normal ovarian tissue and ovarian cancer tissue specimens.

Results

DPI treatment significantly induced apoptosis in both EOC cell lines as evident by increased caspase-3 activity and TUNEL assay. Additionally, both EOC cell lines were found to express NADPH oxidase, HIF-1α, and SOD3, which were highly sensitive to DPI treatment. DPI treatment resulted in reduced NADPH oxidase, SOD3 and HIF-1α levels. Furthermore, ovarian cancer tissues were found to manifest higher NADPH oxidase levels as compared to normal ovarian tissues.

Conclusions

These data suggest that lowering oxidative stress, possibly through the inhibition of NADPH oxidase, induces apoptosis in ovarian cancer cells and may serve as a potential target for cancer therapy.     

Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

Objectives

To examine the effects of combination therapy with metformin and paclitaxel in endometrial cancer cell lines.

Methods

ECC-1 and Ishikawa endometrial cancer cell lines were used. Cell proliferation was assessed after exposure to paclitaxel and metformin. Cell cycle progression was assessed by flow cytometry. hTERT expression was determined by real-time RT-PCR. Western immunoblotting was performed to determine the effect of metformin/paclitaxel on the mTOR pathway.

Results

Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC₅₀ values of 1-5 nM and 5-10 nM for Ishikawa and ECC-1 cells, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with metformin (0.5 mM) resulted in a significant synergistic anti-proliferative effect in both cell lines (Combination Index < 1). Metformin induced G1 arrest in both cell lines. Paclitaxel alone or in combination with metformin resulted in predominantly G2 arrest. Metformin decreased hTERT mRNA expression while paclitaxel alone had no effect on telomerase activity. Metformin stimulated AMPK phosphorylation and decreased phosphorylation of the S6 protein. In contrast, paclitaxel inhibited AMPK phosphorylation in the ECC-1 cell line and induced phosphorylation of S6 in both cell lines. Treatment with metformin and paclitaxel resulted in decreased phosphorylation of S6 in both cell lines but only had an additive effect on AMPK phosphorylation in the ECC-1 cell line.

Conclusions

Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway. This combination may be a promising targeted therapy for endometrial cancer.     

Brain metastases from epithelial ovarian carcinoma: evaluation of prognosis and managements - a Taiwanese Gynecologic Oncology Group (TGOG) study

Objective

To evaluate the characteristics and outcome of patients with brain metastases from epithelial ovarian carcinoma.

Methods

The clinical and pathologic characteristics, treatment and outcome of patients with brain metastases from epithelial ovarian carcinoma were analyzed from eight medical centers in Taiwan under the TGOG (Taiwanese Gynecologic Oncology Group).

Results

A total of 64 patients were recruited in this study. The incidence of brain metastases from epithelial ovarian carcinoma seemed to be increasing in recent years. The median survival from the diagnosis of brain metastases was 8 months (range: 0-72). Prior cancer relapse before the diagnosis of brain metastases, number of brain metastases and multimodal treatment were related to the duration of survival.

Conclusions

The prognosis for patients with brain metastases from epithelial ovarian carcinoma is generally poor. However, clinicians should keep alert to the neurological complaints of ovarian cancer patients and the patients might benefit from aggressive multimodal treatments.     

The opioid growth factor (OGF) and low dose naltrexone (LDN) suppress human ovarian cancer progression in mice

Objective

The opioid growth factor (OGF) and its receptor, OGFr, serve as a tonically active inhibitory axis regulating cell proliferation in normal cells and a variety of cancers, including human ovarian cancer. Blockade of OGF and OGFr with the nonselective opioid receptor antagonist naltrexone (NTX) upregulates expression of OGF and OGFr. Administration of a low dosage of NTX (LDN) blocks endogenous opioids from opioid receptors for a short period of time (4-6 h) each day, providing a window of 18-20 h for the upregulated opioids and receptors to interact. The present study investigated the repercussions of upregulating the OGF-OGFr axis by treatment with OGF or LDN on human ovarian tumorigenesis in vivo.

Methods

Female nude mice were transplanted intraperitoneally with SKOV-3 human ovarian cancer cells and treated on a daily basis with OGF (10 mg/kg), LDN (0.1 mg/kg), or an equivalent volume of vehicle (saline). Tumor burden, as well as DNA synthesis, apoptosis, and angiogenesis was assessed in tumor tissue following 40 days of treatment.

Results

OGF and LDN markedly reduced ovarian tumor burden (tumor nodule number and weight). The mechanism of action was targeted to an inhibition of tumor cell proliferation and angiogenesis; no changes in cell survival were noted.

Conclusions

This study shows that a native opioid pathway can suppress human ovarian cancer in a xenograft model, and provides novel non-toxic therapies for the treatment of this lethal neoplasia.