Zinc induces apoptosis on cervical carcinoma cells by p53-dependent and -independent pathway.

OBJECTIVE: There is evidence that the mineral zinc is involved in the apoptotic cell death of various carcinoma cells. In this study, we aim to determine whether zinc in the form of CIZAR induces apoptosis in cervical carcinoma cells by increasing intracellular zinc concentration. STUDY DESIGN: CaSki and HeLa cervical carcinoma cells and HPV-16 DNA-transformed keratinocyte (CRL2404) were treated with different concentrations of CIZAR. The cell viability test was carried out, the intracellular level of zinc was determined, and apoptosis was confirmed by flow cytometry after propidium iodide (PI) staining and fluorescence microscopy under DAPI staining. The expression of cell-cycle regulators was analyzed by Western blot, including the knock down of p53 and expression of HPV E6 and E7 genes by RT-PCR. RESULTS: Intracellular zinc accumulation induced the down-regulation of E6/E7 proteins through targeting of the specific transcriptional factors in the upstream regulatory region. p53 was induced after CIZAR treatment and p53-dependent apoptosis did not occur after knock down by p53 siRNA. In cervical carcinoma cells, regardless of HPV-infection, CIZAR induces apoptosis by the activation of the p53-independent pathways through the up-regulation of p21waf1, the down-regulation of c-Myc, and by decreasing the Bcl-2/Bax ratio. CONCLUSIONS: CIZAR induces apoptosis not only through the restoration of p53/Rb-dependent pathways in HPV-positive cells, but also through the activation of p53/Rb-independent pathways and the mitochondrial death-signal pathway in cervical carcinoma cells regardless of HPV-infection.

Conservative Chemotherapy in Gestational Trophoblastic Disease: Experience With Etoposide, Methotrexate, and Dactinomycin Chemotherapy.

OBJECTIVE: The goal of this study was to evaluate the efficacy, toxicity, and survival of patients in our institution treated by EMA (etoposide, methotrexate [MTX], and dactinomycin) chemotherapy for 3 groups of patients: ones that had low-risk gestational trophoblastic disease (GTD) that was resistant to MTX (group A), those with high-risk GTD (group B), and the group having low-risk GTD but the cancer being metastatic (group C). METHODS: The medical records of 58 patients who received EMA chemotherapy in groups A, B, and C in the 2000 to 2012 period at St Mary's Hospital were examined. Clinical characteristics, chemotherapy responses, causes of treatment failure, and cases of drug toxicity were analyzed retrospectively. RESULTS: Treatment with the EMA regimen resulted in primary remission in 52 (96%) of 54 patients and resistance in 2 of the patients (3%). In the resistance group, one belonged to group B and was treated with etoposide, MTX, and actinomycin D with cyclophosphamide and vincristine (EMA-EP) and the other belonged to group A and died of refractory disease. World Health Organization (WHO) grade 4 leukocytopenia and thrombocytopenia with the EMA regimen occurred in 6% and 0.4% of the cycles, respectively; the other toxic effects were acceptable and manageable. Median cycles of EMA chemotherapy during the treatment were 7, 8, and 8 in groups A, B, and C, respectively. There was some reduction in total chemo cycle and toxicity, as compared with a previously reported study using the alternative cyclophosphamide and vincristine regimen. Among the EMA treated patients, 1 patient with a second malignancy of breast cancer was documented. In addition, 5 child births for the treated patients were recorded during the follow-up period of mostly 10 years. CONCLUSIONS: The EMA chemotherapy seemed to reduce treatment duration and the relapse rate without increasing the adverse effects in patients with MTX resistance and low-risk GTD, but having confirmed metastatic lesions. Although this study had some limitations regarding the high-risk GTD, our findings will provide a basis for the use of EMA chemotherapy when cyclophosphamide and vincristine is contraindicated due to toxicity.

Large cell neuroendocrine carcinoma of the ovary: a case report and a brief review of the literature.

Large cell neuroendocrine carcinoma (LCNC) of the ovary, or ovarian undifferentiated non-small cell carcinoma of neuroendocrine type, is a rare entity that is frequently associated with ovarian surface epithelial tumors. Few cases have been reported in the literature. LCNC is an aggressive tumor with tendency to present at advanced stages and to cause death after a short postoperative duration. We report three cases of LCNC diagnosed histopathologically. Immunohistochemically, the tumor cells were positive for chromogranin A, NSE, CD56, and pancytokeratin. The patients were treated postoperatively with combination chemotherapy. Due to the rarity of LCNC, the general consensus on standard therapy is not established. Although most patients are at stage I, the biological aggressiveness and poor prognosis of the tumors have been reported in previous reports despite extensive surgery and chemotherapy.

Correlation between the digital cervicography and pathological diagnosis performed at private clinics in Korea.

OBJECTIVE: To evaluate the correlation tendency between abnormal findings of digital cervicography and cervical pathology at private clinics in Korea. METHODS: Abnormal finding of digital cervicography performed at private clinics in Korea between January 1, 2010 and May 31, 2012 were analysed retrospectively. The patient's age, abnormal findings of digital cervicography, cervical cytology, human papillomaviru (HPV) test and cervical pathology were investigated and the rate of agreement between abnormal finding of digital cervicography and cervical pathology results was calculated. Abnormal findings of digital cervicography were divided into 4 categories: atypical, compatible with CIN1, compatible with CIN2/3 and compatible with cancer. RESULTS: The study group was composed of 1547 women with a mean (range) age of 37.4 (14-91 years). The agreement rate between abnormal findings of digital cervicography and cervical pathology was 52.0% in "compatible with CIN1", 78.9% in "compatible with CIN2/3", and 90.2% in "compatible with cancer". CONCLUSIONS: Abnormal findings of digital cervicography were highly concordant with cervical intraepithelial neoplasia (CIN) and cancer examined at outpatient clinics in Korea. Therefore, abnormal interpretations of digital cervicography can be used as an excellent auxiliary technique with cervical cytology for CIN and cancer.

Incidence and risk factors of lower-extremity lymphedema after radical surgery with or without adjuvant radiotherapy in patients with FIGO stage I to stage IIA cervical cancer.

OBJECTIVE: This study aimed to determine the incidence and risk factors of lower-extremity lymphedema (LEL) in women who had radical surgery with or without adjuvant radiotherapy for International Federation of Gynecology and Obstetrics (FIGO) stage I to stage IIA cervical cancer. METHODS: The medical records were reviewed retrospectively on patients with histologically confirmed FIGO stage I to IIA cervical cancer. Lower-extremity lymphedema-related medical problems such as peripheral vascular disease, congestive heart failure, or chronic renal disease were excluded. A logistic regression analysis was used to examine the relationship between variable clinical characteristics and development of LEL. RESULTS: We evaluated 707 patients. Of the 707 patients evaluated, we excluded 92 patients who had received radiotherapy as the initial therapy and 19 patients with LEL related to medical problems. Seventy-five patients (12.6%) developed LEL. The incidence was high in patients with adjuvant radiotherapy (odds ratio, 3.47; 95% confidence interval, 2.086-5.788; P = 0.000), with 78.7% of the patients with LEL having developed the condition within 3 years after initial treatment. CONCLUSIONS: Adjuvant radiotherapy was significantly associated with development of LEL in women who had undergone radical surgery with lymphadenectomy for FIGO stage I to stage IIA cervical cancer. The possibility for the occurrence of LEL must be fully explained before treatment and patients should be provided with the appropriate preventive education. Further prospective studies are needed to confirm the incidence and risk factors for LEL.

A pilot study to investigate the treatment of cervical human papillomavirus infection with zinc-citrate compound (CIZAR®).

OBJECTIVE: In the present study the potential therapeutic effects of zinc-citrate compound (CIZAR®) in women infected with high-risk human papillomavirus (HR-HPV) was investigated. METHODS: A total of 194 women diagnosed with HR-HPV infection using the Hybrid capture (HC) II assay with no evidence of high grade squamous intraepithelial lesions (HSIL) or worse by Pap smear and colposcopy were enrolled. Among them, 76 women were treated by twice weekly self administered intra-vaginal infusion of 0.5 mM zinc citrate solution containing CIZAR® for 12 weeks and were evaluated for clearance of the HR-HPV infection compared to 118 women without treatment (Control group). RESULTS: The 12 weeks zinc citrate solution treatment resulted in the elimination of HR-HPV in 49/76 (64.47%) patients compared to the spontaneous clearance of 15.25% (18/118) in the control group (p=0.000). By logistic regression analysis, the 12 week zinc citrate solution treatment reduced the risk of persistent HR-HPV infection significantly (OR 0.079; 95% CI 0.039-0.165; p=0.000). CONCLUSION: The results of this study showed for the first time that treatment with intra-vaginal infusion of a zinc-citrate compound (CIZAR®) can result in elimination of HR-HPV infection from the uterine cervix.

ERCC1 (excision repair cross-complementation group 1) expression as a predictor for response of neoadjuvant chemotherapy for FIGO stage 2B uterine cervix cancer.

OBJECTIVE: Platinum-based neoadjuvant chemotherapy for locally advanced cervical cancer has some benefits for patients responding to chemotherapy. However, no validated clinical or biologic predictor of response to this chemotherapy has been identified to date. METHODS: We employ immunohistochemical analysis to determine the expression patterns of the excision repair cross-complementation group1 (ERCC1) protein in pre-treatment cervical biopsy tissue. In total, 43 stage IIB patients had been enrolled in a previous etoposide and cisplatin neoadjuvant phase II clinical trial, allowing comparison of the effects of cisplatin-based neoadjuvant chemotherapy on response in relation to ERCC1 expression. RESULTS: Among the 43 patients studied, 34 (79.1%) were positive and 9 (20.9%) were negative for ERCC1. Response to chemotherapy (according to RECIST criteria) was observed in all patients with negative ERCC1 expression. In logistic regression analysis, ERCC1 negativity continued to be an independent predictor for responsiveness to neoadjuvant chemotherapy (p=0.021). Among the pretreatment factors, low ERCC1 expression was a significant prognostic factor of disease-free survival in multivariate analysis (p=0.046). CONCLUSIONS: The ERCC1 expression patterns in pretreatment specimens may thus facilitate the prediction of responses to cisplatin-based NAC. We propose that patients expressing low levels of ERCC1 derive the most benefit from cisplatin-based NAC.

Clinical analysis of intra-operative frozen section proven borderline tumors of the ovary.

OBJECTIVE: We have assessed the accuracy of frozen section diagnosis and the outcomes of misdiagnosis in borderline tumors of the ovary (BTO) according to frozen section. METHODS: All pathology reports with BTO in both frozen and permanent section analyses between 1994 and 2008 at Seoul St. Mary's Hospital were reviewed. Frozen section diagnosis and permanent section histology reports were compared. Logistic regression models were conducted to evaluate the correlation of patient and tumor characteristics with diagnostic accuracy. The clinical outcomes of misdiagnosis were evaluated. RESULTS: Agreement between frozen section diagnosis and permanent histology was observed in 63 of 101 patients (62.4%). Among the 76 patients with frozen section proven BTO, under-diagnosis and over-diagnosis occurred in 8 of 76 (10.5%) and 5 of 76 patients (6.6%), respectively. Mean diameter of under-diagnosed tumor was larger than matched BTO (21.0+/-11.4 vs. 13.7+/-7.1; p=0.021). Tumor size 20 cm was determined as the optimal cut-off for under-diagnosis (50% sensitivity, 87.3% specificity). Among 8 under-diagnosed patients, no patient relapsed. Among 5 over-diagnosed patients, 2 patients < 35 years of age had fertility-preserving surgery. CONCLUSION: Although frozen section diagnosis is an important and reliable tool in the clinical management of patients with ovarian tumors, over-diagnosis and under-diagnosis are relatively frequent in frozen proven BTO. Surgical decision-making for BTO based on frozen section diagnosis should be done carefully, especially in large tumors.

Neoadjuvant cisplatin and etoposide followed by radical hysterectomy for stage 1B-2B cervical cancer.

OBJECTIVES: We aimed to determine the efficacy and feasibility of neoadjuvant chemotherapy (NACT) using cisplatin and etoposide in patients with locally advanced cervical cancer. METHODS: Previously untreated patients with histologically confirmed stage 1B-2B cervical cancer were treated with three courses of NACT (60 mg/m2 cisplatin on days 1 and 2 plus 100 mg/m2 etoposide on day 1) every 10 days. NACT was followed within 2-3 weeks by radical hysterectomy with lymph node dissection. RESULTS: From 1999 to 2004, 112 patients were enrolled and 99 patients were evaluable. All eligible patients had radical surgery after NACT. Hematologic toxicity was the most common side effect, and the level of toxicity was acceptable. The overall pathologic response rate was 69.7% (69/99). The median follow-up period was 49 months, and the 5-year overall and progression-free survival rates were 88.1% and 60.5%, respectively. CONCLUSION: NACT with cisplatin and etoposide for patients with locally advanced cervical cancer is promising and has an acceptable toxicity profile. The regimen timing (every 10 days) did not delay the optimal time for radical treatment.

Adjuvant therapy in high-risk early endometrial carcinoma: a retrospective analysis of 46 cases.

OBJECTIVE: We assessed the prognostic factors and the efficacy of adjuvant therapy and reviewed randomized studies carried out on patients receiving adjuvant therapy with early endometrial carcinoma. METHODS: One hundred and five patients that received primary surgical treatment for stage IB, IC and II endometrial cancer were enrolled in this study. The clinical outcomes were compared among the patients with variable prognostic factors and adjuvant treatments. RESULTS: One hundred and five patients fulfilled the eligibility criteria and 46 patients (43.8%) underwent adjuvant therapy. Disease recurrence occurred in nine patients within a median time of 24 months. Cervical involvement was an independent prognostic factor for the disease-free survival rates. Eight of 16 patients with FIGO stage II disease received adjuvant chemotherapy consisting of cisplatin and etoposide (or cyclophosphamide) or combined chemoradiation. The 5-year disease-free survival rate for these patients was 87.5%, a value significantly higher than for patients that received radiation therapy alone (30%). CONCLUSION: Adjuvant chemotherapy or combination chemo-radiotherapy might be superior to radiation therapy alone in high-risk early endometrial cancer patients.

Clinical analysis of synchronous primary neoplasms of the female reproductive tract.

OBJECTIVES: In this study, a histopathologic review of synchronous primary neoplasms including gynecologic malignancies is presented, and the possible correlation among discrete tumor subsets, natural history, and survival is evaluated. METHODS: Between the years 2000 and 2005, 20 patients suffering from synchronous primary cancers of gynecologic malignancy were identified. Clinical and pathologic information was obtained from medical records. Kaplan-Meier survival analyses were conducted. RESULTS: Patients with synchronous primary malignancies constituted 0.63% of all genital malignancies. The most frequently observed synchronous neoplasm was ovarian cancer coexistent with endometrial cancer (40%). The mean age of patients suffering from synchronous ovarian and endometrial cancer was 45.2 years. All patients with synchronous primary genital malignancies underwent hysterectomy with bilateral salpingo-oophorectomy and/or adjuvant therapy. The mean duration of survival was 57 months (S.E.: 10.0; 95% confidence interval: 37-77). CONCLUSION: Patients suffering from primary genital malignancies are sometimes co-afflicted with other primary cancers. Synchronous ovarian and endometrial cancer constitutes the most common of these cases, and is detected at a relatively early age, with generally favorable prognoses.

Management of high-risk hydatidiform mole and persistent gestational trophoblastic neoplasia: the Korean experience.

OBJECTIVE: To test the efficacy of a new scoring system to differentiate high-risk hydatidiform mole (H-mole) and initiate early selective postmolar chemotherapy. STUDY DESIGN: According to Kim's scoring system, 262 patients were identified as high-risk H-mole patients. Fifty (19.1%) received early chemotherapy, and the rest constituted the control group. Salvage therapy with etoposide, methotrexate, actinomycin D/etoposide, cisplatin (EMA/EP) and taxol, cisplatin/taxol, etoposide (TP/TE) was applied in 21 cases of ultra-high-risk GTT. RESULTS: None of the 50 cases in the early chemotherapy group progressed to persistent GTT. However, 58.9% in the control group developed GTT with 8.0% drug resistance. Of those receiving salvage therapy in the 21 ultra-high-risk GTT cases resistant to EMA/CO, 10 of 14 (71%) receiving EMA/EP and 4 of 7 (57.1%) receiving TP/TE achieved remission. CONCLUSION: Early postmolar chemotherapy for high-risk H-mole is effective in preventing progression to persistent GTT and treatment failure. Ultra-high-risk GTT should be approached with multimodal treatment, including EMA/EP and TP/TE regimens.

Antiproliferative and apoptotic effects of zinc-citrate compound (CIZAR(R)) on human epithelial ovarian cancer cell line, OVCAR-3.

OBJECTIVE: Zinc inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. The intracellular concentration of zinc and its dynamic changes are critically important in cell biology. We investigated the effects of zinc-citrate compound (CIZAR) on normal human ovarian epithelial cells (NOSE) and human epithelial ovarian cancer cell line, OVCAR-3. METHODS: To investigate the potential effect of CIZAR on cell growth and survival, cells were treated with different doses and exposed to different times. Intracellular concentration of zinc was measured by colorimetric assay. Mitochondrial aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, and morphological analysis were done to investigate cytotoxic effects of CIZAR. Molecular mechanism of cell death was investigated by p53, Bcl-xL, Bcl-2, Bax protein, activity of caspase-3 and -12, and activity of telomerase. RESULTS: CIZAR-induced zinc accumulation in OVCAR-3 cells was higher than that in NOSE cells. CIZAR(R) treatment resulted in a time- and dose-dependent decrease in cell number in OVCAR-3 cells in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells within 4 h exposure to CIZAR but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering, and morphological analysis indicated apoptosis in OVCAR-3 cells but not in NOSE cells. CIZAR increased the expression of p21(waf1) which is a part of p53-independent pathway and induced reduction of telomerase activity. CIZAR reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR induced apoptosis of OVCAR-3 cells by activation of caspase-12 and caspase-3 pathway. CONCLUSIONS: Exposure to CIZAR induces apoptosis in OVCAR-3 cells which accumulate high intracellular levels of zinc, but not in NOSE cells, which do not accumulate high levels of zinc. CIZAR(R) prevents the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induces apoptosis by induction of proapoptotic gene (Bax), repression of antiapoptotic genes (Bcl-2, Bcl-xL), and consequently activation of caspase-3. CIZAR also induced activation of caspase-12. The CIZAR will offer new window in prevention and treatment of epithelial ovarian cancer.

Necrotic cell death of ovarian adenocarcinoma caused by seminal plasma.

OBJECTIVE: From the knowledge of risk factors of epithelial ovarian cancer, we deduced a hypothesis that human seminal plasma (HSP) has a preventive role in the development of epithelial ovarian cancer. To examine whether HSP directly influences the growth of ovarian cancer, we have investigated the in vitro and in vivo effect of HSP on ovarian adenocarcinoma cell lines (SK-OV-3 and OVCAR-3) in comparison with its effects on normal ovarian surface epithelial cells (NOSE). METHODS: Cell viability was determined by MTT assay. Cytotoxic effect was evaluated by flow cytometry analysis, by DNA laddering, and by morphological analysis. In vivo therapeutic effect of HSP was evaluated by the subcutaneous inoculation of SK-OV-3 cells in nude mice (BALB-c) model. RESULTS: HSP at a final concentration of 1:50 induced a time- and dose-dependent inhibition of SK-OV-3 and OVCAR-3 growth, whereas NOSE was not affected. Flow cytometric analysis, DNA laddering, and morphological analysis indicated that HSP induced necrosis, rather than apoptosis, of both ovarian carcinoma cell lines. In in vivo experiment that used the nude mice (Balb-C) with tumor inoculation of SK-OV-3 cells, HSP induced necrosis of tumor with no detectable toxic effects on the major organs. CONCLUSION: These results show that HSP inhibits the growth and induces the necrosis of epithelial ovarian cancer cells and suggests that one or more components of HSP may provide a scientific basis for preventing epithelial ovarian cancer.

Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice.

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.

Sentinel node biopsy as an indicator for pelvic nodes dissection in early stage cervical cancer.

The purpose of this study was to investigate the feasibility of sentinel node frozen biopsy to minimize the extensive pelvic lymph nodes dissection in early stage cervical cancer patients on the basis that the risk of skip metastasis to the paraaortic area is negligible. Twenty-six patients with early stage cervical cancer were enrolled in this study. Technetium-99m colloid albumin (Tc(99m)) was injected intradermally around the tumor for allowing preoperative lymphoscintigraphy and intraoperative hand-held gama probe detection of seninel nodes. For visual detection, isosulfan blue dye was injected into the peritumoral sites before peritoneal opening. Postoperative morbidity and negative predictive value were the endpoints of this study. The 26 patients, ranging in age from 32 to 71 yr, underwent intraoperative sentinel nodes mapping. All the patients underwent complete pelvic lymph nodes dissection including para-aortic nodes. There was one case with positive non-sentinel nodes despite the negative sentinel node by frozen biopsy (negative predictive value, 95.2%). This new technique of sentinel node mapping is safe and simple to perform. Further clinical trials using the combination of Tc(99m) and isosulfan blue dye are warranted and this technique will make a true advance for less aggressive management of patients with early stage cervical cancer.