VEGF- and LPA-induced telomerase in human ovarian cancer cells is Sp1-dependent

OBJECTIVE: Both vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) are secreted by ovarian cancer cells and are known to promote cancer cell growth though the exact mechanism(s) are not completely understood. Since telomerase, a ribonucleprotein expressed in 95% of ovarian cancers, plays an important role in cellular immortalization, growth, and tumor progression, we examined whether telomerase is a molecular target of LPA and VEGF in ovarian cancer. METHODS: Telomerase-positive ovarian carcinoma cell lines PA-1, SW 626, and one telomerase-negative, non-tumorigenic SV40 large-T antigen-transfected human ovarian surface epithelial (IOSE) cell line, FHIOSE 118, derived from normal ovarian surface epithelium were cultured with and without VEGF and LPA for 4 h and 24 h, respectively. Telomerase PCR-ELISA, RT-PCR, VEGF ELISA and luciferase assays were performed to determine the effect of VEGF and LPA on telomerase activity in ovarian cancer cells. Western blot analyses were used to examine the signaling pathway involved in telomerase regulation by VEGF and LPA. RESULTS: We report that: (1) both VEGF and LPA upregulate telomerase activity; (2) LPA induction of telomerase activity is VEGF-dependent; (3) VEGF and LPA induction of telomerase activity is ERK 1/2-dependent; and (4) Sp1 binding sites within the proximal 976- to 378-bp regions of the hTERT promoter are essential for VEGF- and LPA-induced hTERT promoter activity. CONCLUSION: Consequently, these data show the novel finding that VEGF can regulate telomerase activity in non-endothelial cells and that telomerase appears to be a novel molecular target of LPA.     

端粒酶活性与卵巢上皮性肿瘤临床病理因素相关性分析

目的 探讨端粒酶在上皮性卵巢肿瘤发生发展中的作用,评价端粒酶作为卵巢癌诊断及预后指标的价值。方法 采用端粒酶PCR-ELISA法对25例卵巢上皮性肿瘤包括8例良性(5例浆液性、3例粘液性)、3例交界性(浆液性)和14例恶性(8例浆液性,4例粘液性,2例内膜样),6例正常卵巢表面上皮进行端粒酶活性定量检测。结果 2例良性、2例交界性和12例恶性卵巢上皮性肿瘤及1例正常上皮存在端粒酶活性,端粒酶活性的吸光度(A)平均值分别为0.080±0.070、0.408±0.208、1.659±0.930和0.086±0.060,统计学分析表明恶性卵巢肿瘤中端粒酶活性明显高于良性、交界性肿瘤和正常卵巢组织,端粒酶活性和卵巢上皮性肿瘤组织学类型、临床分期无关;而与肿瘤的分化程度呈负相关。结论 研究结果初步证明端粒酶活性增高在卵巢癌的无限增殖中是一重要环节,并提示端粒酶表达有可能成为卵巢癌早期诊断及判断预后的肿瘤标志物。端粒酶PCR-ELISA法具有定性又定量、特异性较高、敏感性强、简易快速、可避免同位素污染等优点。     

Women with high telomerase activity in luteinised granulosa cells have a higher pregnancy rate during in vitro fertilisation treatment

Objective

To study the effect of telomerase activity (TA) in human luteinised granulosa cells (GCs) on the outcome of in vitro fertilisation treatment.

Methods

Fifty-six women, aged 23 to 39 years, were enrolled and divided into four groups according to their levels of TA.

Results

Seventeen cases in group A exhibited nondetectable TA, 16 cases in group B expressed low levels of TA (between 0.1 and 0.65 OD × mm), 14 cases in group C expressed moderate TA levels (between 0.66 and 1.00 OD × mm) and 9 cases in group D expressed high levels of TA (more than 1.00 OD × mm). The level of total serum testosterone (T) was significantly higher in groups C and D than in group A (1.43 ± 0.10 vs. 1.08 ± 0.11 nmol/L, P < 0.030 and 1.56 ± 0.08 vs. 1.08 ± 0.11 nmol/L, P < 0.005, respectively). The TA level was positively correlated with T (r = 0.291, P < 0.011). No obvious differences were observed in rates of fertilisation, cleavage, mature oocyte formation or good-quality embryos among the groups. The patients in group D exhibited the highest rates of embryo implantation and clinical pregnancy (with rates of 52.63% and 77.78%, respectively, compared with 18.92% and 29.41% in group A, 25.71% and 37.50% in group B and 48% and 50% in group C, with P < 0.018 and P = 0.112, respectively). The patients in group D also had a greater likelihood of becoming pregnant than those in group A (OR: 9.703, P < 0.023), group B (OR: 14.765, P < 0.009) or group C (OR: 5.560, P = 0.103).

Conclusions

Luteinised GCs have a certain potential for proliferation and TA of luteinised GCs may predict the clinical outcomes of IVF treatment. Some unknown regulatory mechanisms between TA and T should be studied in further trials.     

黄素化颗粒细胞端粒酶的表达及与卵巢功能关系的初步研究

目的　了解端粒酶在人卵巢黄素化颗粒细胞的表达 ,及与卵巢生殖功能的关系。方法　收集 2 2例行体外受精 胚胎移植或卵胞浆单精子显微注射患者的卵巢黄素化颗粒细胞 ,采用原位杂交法及端粒末端重复序列扩增法 ,分别检测人端粒酶催化亚单位 (hTERT)mRNA及端粒酶活性。结果　黄素化颗粒细胞普遍表达hTERTmRNA ,但其表达强弱不一。 2 2份卵巢黄素化颗粒细胞标本中 ,16份 (73% )有端粒酶活性 ,与血清基础促卵泡激素水平呈负相关 (P <0 .0 1) ,且随年龄增长呈下降趋势。结论　端粒酶可能对维持卵巢功能起重要作用 ,卵巢功能下降可能与卵巢颗粒细胞端粒酶活性降低有关     

The impact of novel retinoids in combination with platinum chemotherapy on ovarian cancer stem cells

Objective

Retinoids are important modulators of cell growth, differentiation, and proliferation. 9cUAB30, 9cUAB124, and 9cUAB130 are three novel retinoid compounds that show cytotoxic effects in other malignancies. We evaluated these novel retinoids in combination with chemotherapy against ovarian cancer stem cells (CSCs) in vitro and in an ex vivo model.

Methods

A2780 cells were plated in 96-well plates and treated with retinoid, carboplatin, or combination therapy. Cell viability was evaluated using ATPLite assay. The A2780 cell line was also analyzed for CSCs by evaluating ALDH activity using flow cytometry. A2780 cells treated ex vivo with retinoids and chemotherapy were injected into the flank of athymic nude mice in order to evaluate subsequent tumor initiating capacity.

Results

A2780 cells were sensitive to treatment with retinoids and carboplatin. The best treatment resulted from the combination of retinoid 9cUAB130 and carboplatin. Untreated A2780 cells demonstrated ALDH activity in 3.3% of the cell population. Carboplatin treatment enriched ALDH activity to 27.3%, while 9cUAB130 ± carboplatin maintained the ALDH positive levels similar to untreated controls (2.3% and 6.7%, respectively). Similar results were found in tumorsphere-forming conditions. Flank injections of ex vivo treated A2780 cells resulted in 4/4 mice developing tumors at 40 days in the untreated group, while 0/4 tumors developed in the 9cUAB130 and carboplatin treated group.

Conclusion

Combination treatment with carboplatin and retinoids reduced cell-viability, reduced CSC marker expression, and inhibited tumorigenicity, making it a more effective treatment when compared with carboplatin alone.     

Alteration of telomerase activity in ovarian cancer after chemotherapy

To elucidate the relationship between telomerase activity and chemosensitivity in epithelial ovarian cancer, telomerase activity and telomere length (TRF) were examined before and after chemotherapy. Of 21 patients, 9 patients responded to chemotherapy and 12 did not. The positivity of telomerase activity did not significantly differ between responders and nonresponders. There were no differences in the mean length and the distribution of TRF between the two groups. Those distributions became narrow after chemotherapy in both groups. Seven nonresponders (58.3%) exhibited an increase in telomerase activity after chemotherapy but none of the responders showed an increase in activity. Telomerase activity may relate to chemosensitivity in epithelial ovarian cancer.     

Predictive value of circulating tumor cells (CTCs) in newly-diagnosed and recurrent ovarian cancer patients

Objective

To determine whether circulating tumor cells (CTCs), as detected and enumerated by the Veridex CellSearch^TM system, could predict for clinical outcomes in women with newly diagnosed or recurrent epithelial ovarian cancer.

Methods

Serial measurements of CTC s and paired serum CA125 were collected in a series of 78 women with newly diagnosed or recurrent ovarian cancer seen at our institution over a period of 15 months. Clinical data were abstracted from patient medical records. CTCs were detected and enumerated by the CellSearch^TM protocol, and patients were divided into CTC negative (< 2 CTCs) or positive (≥ 2 CTCs) groups. CTC groups were correlated with clinical characteristics and outcomes. A longitudinal analysis of CTC change and CA125 trends was also performed.

Results

At least one CTC was isolated from the peripheral blood of over 80% of the women participating in this study, with a range from 0 to 8. No correlations were observed between CTC numbers and clinical characteristics or outcomes. Although both serum CA125 and CTC number exhibited an overall significant decreasing trend over time, there was no correlation observed between change in CTCs and CA125.

Conclusion

Using the FDA-approved CellSearch^TM system, CTCs can be isolated from women with newly diagnosed or recurrent ovarian cancer. However, CTC numbers do not significantly correlate with clinical characteristics or patient outcomes. Future studies should focus on phenotypic characterization of CTCs and whether different isolation protocols yield a higher number of CTCs or add prognostic value.     

Quality of life evaluations in patients with ovarian cancer during chemotherapy treatment

OBJECTIVES: To examine the impact of treatment- and disease-related factors on the quality of life of patients with ovarian cancers undergoing chemotherapy. PATIENTS AND METHODS: Over 18 months period, all patients with ovarian cancer receiving chemotherapy at the Saskatoon Cancer Center were recruited. The Functional Assessment of Cancer Therapy-Ovarian (FACT-O) questionnaire was used to assess patients' quality of life before each chemotherapy cycle. Platinum-based chemotherapy was used initially or in patients with a platinum-free interval of more than 6 months in a recurrence setting. After progression on the platinum-based regimens, liposomal doxorubicin, topotecan, and cisplatinum/etoposide were used as salvage chemotherapy pending on drug availability and convenience of administration to patients. Regression analysis was used to identify significant disease and treatment-related factors that can significantly affect patients' quality of life measures. RESULTS: Seventy-two patients participated in the study providing 270 separate observations. The mean age was 57.81 years with a standard deviation of 13.40. The median duration of chemotherapy-free interval for patients with recurrent disease was 7 months. All patients had stage 3 or 4 disease. About half (52.2%) of the patients had optimal surgical resection with small (<1 cm) residual cancer masses before primary adjuvant chemotherapy. Seventy percent of the patients had either a first diagnosis or a first recurrence of cancer with the other 30% previously treated with two or more chemotherapy regimens. Sixty-two percent had an initial complete response to platinum-based chemotherapy. Multivariate regression analysis showed the use of topotecan or cisplatinum/etoposide, patients' poor responses to chemotherapy, experience with two or more previous line of chemotherapy treatment, and younger ages were significant predictors of poor quality of life during chemotherapy. CONCLUSION: There were significant differences in side effects of commonly used chemotherapy regimens on patients' quality of life. Quality of life assessments should be routinely incorporated in selecting specific chemotherapy to be used. Future research should be carried out to identify the best strategies to further integrate the results of quality of life assessments in cancer treatment protocols and to examine the long-term effects of cancer and its treatment on patients and their families.     

芹菜素抑制人卵巢癌CAOV3细胞增殖的研究

目的探讨芹菜素对人卵巢癌CAOV3细胞增殖的抑制作用。方法2006年10月至12月在中国医科大学附属第一医院肿瘤研究所应用四甲基偶氮唑蓝（MTT）比色法检测芹菜素对CAOV3细胞增殖的抑制作用，流式细胞仪技术分析芹菜素对CAOV3细胞增殖周期的影响。结果20—160μmol／L芹菜素均能抑制CAOV3细胞的生长，且呈明显的时间、剂量依赖性关系。细胞周期分析显示芹菜素组G2／M期细胞比例明显增多，呈量效关系。结论芹菜素抑制CAOV3细胞增殖，可能通过使CAOV3细胞停滞在G2／M期而诱导其凋亡。     

Circulating and disseminated tumor cells in ovarian cancer: A systematic review

Objectives

Detecting circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in the bone marrow of cancer patients has proven feasible and of prognostic value in different neoplasms. However, the clinical significance of CTCs and DTCs in ovarian cancer and its association with outcome remains unclear.

Methods

A literature search in PubMed was performed from January 2000 to December 2013 for studies evaluating CTCs and/or DTCs and its association with clinicopathological characteristics and clinical outcome in ovarian cancer. The main outcome measures were progression-free survival (PFS) and overall survival (OS).

Results

Fourteen studies met the inclusion criteria. Median study size was 84 patients (range 43–216). Median follow-up was 19 months (range 5–52). Most studies were small case series (n < 100; studies; 71%). The majority of studies used an immunophenotyping approach to identify CTCs and/or DTCs, but only 3 studies (21%) used the FDA-approved Cell Search method. Despite the differences in methodology among studies the presence of CTCs and DTCs tended to be associated with higher baseline CA-125 serum levels, higher odds of residual disease after surgery, and worse survival in ovarian cancer across studies. No consistent intra-patient correlation was observed between DTCs detected in the bone marrow and CTCs detected in the blood.

Conclusions

The presence of CTCs and DTCs is associated with adverse clinicopathological characteristics and poor clinical outcomes in ovarian cancer patients. Its implementation as a valuable prognostic tool in the clinical setting requires uniform methodology and prospective validation.     

Factors associated with delivery of neoadjuvant chemotherapy in women with advanced stage ovarian cancer

Purpose

To identify clinical and non-clinical factors associated with utilization of primary cytoreductive surgery (PCS) or neoadjuvant chemotherapy (NACT) in women with advanced stage epithelial ovarian cancer (EOC).

Neoadjuvant chemotherapy in the Medicare cohort with advanced ovarian cancer

Objective

The value of neoadjuvant chemotherapy (NAC) for the treatment of advanced ovarian cancer has yet to be determined. While NAC may facilitate and simplify complete cytoreduction and reduce the risk of surgery, the delay of surgery related to NAC needs to be balanced against any potential benefit.

Methods

Surveillance, Epidemiology and End-Results (SEER) data linked to Medicare claims were used to identify 6844 women with treated stage III/IV epithelial ovarian cancer (1995-2005). Patients were classified by primary treatment (surgery (PDS) or chemotherapy), and the primary chemotherapy group was characterized as having NAC or palliative chemotherapy (PC) based on whether there was documentation that surgery was recommended. We compared surgical complications and survival between the groups.

Results

4827 (71%) of women were treated with PDS, 958 received NAC (14%) and 1059 (15%) had PC. Only 577 (60%) of women with NAC underwent surgery and they had fewer ostomies (8.5% vs. 19.2%, p < 0.001) and fewer infections, gastrointestinal and pulmonary complications than PDS (all p < 0.01). Comparing NAC to PDS there was a 16% increase in the risk of death at 2 years (RR 1.16, 95%CI 1.01-1.34) for women with stage III disease and a 15% reduction in the risk for women with stage IV disease (RR 0.85, 95%CI 0.73-0.99).

Conclusions

NAC followed by surgery was associated with fewer surgical complications than PDS. The direction and magnitude of the difference in survival between women receiving NAC and those receiving PDS differed according to the stage of disease and follow up time.     

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.     

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells

Objectives

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells.

The telomerase activity and expression of hTERT gene can serve as indicators in the anti-cancer treatment of human ovarian cancer

OBJECTIVE: The aim of this study was to evaluate the clinicopathological significance of telomerase activity and expression of hTERT gene in human ovarian cancer. The potential value of them as indicators for chemotherapy in ovarian cancer cells was also studied. MATERIALS AND METHODS: A total of 73 samples and ovarian cancer cell lines HO-8910 and COC1 were studied. Telomerase activity was detected by PCR-TRAP-ELISA assay and the expression of the hTERT mRNA was analyzed by semi-quantitative RT-PCR. Alteration of the telomerase activity and hTERT mRNA were also analyzed in the ovarian cancer cells treated with different concentration and different time of cisplatin. Cytogenetic analysis was performed to compare the telomere status in the OH-8910 cells pre- and post-cisplatin treatment. The associations between these two markers and cisplatin induced-apoptosis were respectively analyzed in COC1 cells by the flow cytometry. RESULTS: Telomerase activity are highly increased in malignancy (0.795+/-0.168(A450-655 nm)) than borderline (0.389+/-0.174(A450-655 nm)), benign tumors (0.236+/-0.102(A450-655 nm)) and normal ovary (0.213+/-0.070(A450-655 nm)) (p < 0.05). Twenty samples showed detectable levels of hTERT. The hTERT gene positive lesion showed significantly higher telomerase activity than negative (p = 0.004). There is a significant correlation between the telomerase activity and expression of hTERT (r = 0.921). Both telomerase activity and expression of hTERT can reflect the chemotherapeutic effect of cisplatin in a time-dependent and dose-dependent manner. Treatment with 10 microM cisplatin, the hTERT mRNA decreased after 12h and completely disappeared after 48 h, whereas the telomerase activity did not decrease until 24h. Results from cytogenetic analysis and flow cytometry assay confirmed that the alterations of these two markers are associated with the anti-cancer treatment of cisplatin. CONCLUSION: Expression of hTERT gene is rate-limiting with the activation of telomerase. Both of they may be useful in the predicting of chemotherapeutic effect in ovarian cancer.     

Monoclonal antibody-based immunotherapy of ovarian cancer: Targeting ovarian cancer cells with the B7-H3-specific mAb 376.96

Objective

The high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs.

Methods

Eight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs.

Results

The mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs.

Conclusion

The mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted.