Soshi Kusunoki

The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells

GOALS We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial-mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells. METHOD We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX, BCL2, LEF1, cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin. RESULTS We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells. CONCLUSION This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs.

Sodium butyrate inhibits the self-renewal capacity of endometrial tumor side-population cells by inducing a DNA damage response

We previously isolated side-population (SP) cells from a human endometrial cancer cell line, Hec1, and determined that Hec1-SP cells have cancer stem–like cell features. In this study, we isolated SP cells and non-SP (NSP) cells derived from a rat endometrial cell line expressing human [12Val] KRAS (RK12V cells) and determined the SP phenotype. RK12V-SP cells showed self-renewal capacity, the potential to develop into stromal cells, reduced expression levels of differentiation markers, long-term proliferating capacity in cultures, and enhanced tumorigenicity, indicating that RK12V-SP cells have cancer stem–like cell features. RK12V-SP cells also display higher resistance to conventional chemotherapeutic drugs. In contrast, treatment with a histone deacetylases (HDAC) inhibitor, sodium butyrate (NaB), reduced self-renewal capacity and completely suppressed colony formation of RK12V-SP cells in a soft agar. The levels of intracellular reactive oxygen species (ROS) and the number of γH2AX foci were increased by NaB treatment of both RK12V-SP cells and RK12V-NSP cells. The expression levels of γH2AX, p21, p27, and phospho-p38 mitogen-activated protein kinase were enhanced in RK12V-SP cells compared with RK12V-NSP cells. These results imply that treatment with NaB induced production of intracellular ROS and DNA damage in both RK12V-SP and RK12V-NSP cells. Following NaB treatment, DNA damage response signals were enhanced more in RK12V-SP cells than in RK12V-NSP cells. This is the first article on an inhibitory effect of NaB on proliferation of endometrial cancer stem–like cells. HDAC inhibitors may represent an attractive antitumor therapy based upon their inhibitory effects on cancer stem–like cells. Mol Cancer Ther; 10(8); 1430–9. ©2011 AACR.

Laparoscopic technique of para-aortic lymph node dissection: A comparison of the different approaches to trans- versus extraperitoneal para-aortic lymphadenectomy

Since Dr Dargent first reported endoscopic surgery using retroperitoneal pelvicoscopy to perform pelvic lymph node sampling in 1987, many literature reviews on the safety and feasibility of laparoscopic staging surgery of gynecologic malignancies have been published. However, the procedure of laparoscopic lymphadenectomy is more difficult to perform due to the limited surgical space and associated technical problems. Especially in the para-aortic lymphadenectomy procedure, there are many barriers to overcome in the surgical field, learning curve, and technique. We present a review of lymphadenectomy, especially para-aortic lymphadenectomy.

Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo

The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial–mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

Drug-induced aortitis in a patient with ovarian cancer treated with bevacizumab combination therapy

OBJECTIVE To review and evaluate drug-induced vasculitis, which is an extremely rare complication of chemotherapy. CASE REPORT A 47-year-old woman with ovarian cancer developed aortitis during bevacizumab combination chemotherapy. Contract-enhanced CT showed concentric thickening of the descending aorta. Antibiotics were administered, but a repeat CE-CT scan showed no resolution of the aortitis. To treat the aortitis, she was started on oral prednisolone. A subsequent CE-CT scan showed no signs of aortitis. She was thus re-started on a modified chemotherapy regimen. CONCLUSION Aortitis should be considered in patients receiving bevacizumab combination therapy who develop persistent fever and upper-abdominal pain. Contrast-enhanced CT is useful for detecting drug-induced aortitis.

Long‐term efficacy and safety of aromatase inhibitor use for leiomyomatosis peritonealis disseminata

Leiomyomatosis peritonealis disseminata is a rare disease characterized by pelvic smooth‐muscle nodules of various sizes. It is sometimes misdiagnosed as ovarian or peritoneal carcinoma metastasis; therefore, surgical excision for pathological diagnosis is required. Treatment options include bilateral salpingo‐oophorectomy (BSO), gonadotrophin‐releasing hormone agonist therapy, and aromatase inhibitor therapy. All of these suppress estrogen levels, but a standard treatment has not been established. A 40‐year‐old woman had multiple pelvic tumors, suspicious for ovarian cancer. She underwent laparotomy, where frozen sections of the nodules revealed leiomyomatosis peritonealis disseminata. After she completed gonadotrophin‐releasing hormone agonist therapy, we performed a total abdominal hysterectomy and BSO with residual‐nodule resection, but the nodules recurred 6 months after surgery. We then started letrozole, and 3 years have now elapsed without nodule enlargement or development of new lesions. The long‐term use of aromatase inhibitor therapy is thought to be effective and safe for patients with recurrence after BSO.

Polycystic ovary syndrome with asynchronous bilateral adnexal torsion in a natural cycle

Cases involving polycystic ovaries (PCOs) with adnexal torsion in a natural cycle without ovulation induction are rare, and no reports of such cases have described asynchronous bilateral adnexal torsion. Here, we report a case of PCO syndrome (PCOS) with asynchronous bilateral adnexal torsion in a natural cycle. The patient was a 37-year-old woman with a history of 2 gravidas 1 para. Her primary complaint was left lower abdominal pain. Ultrasonography and MRI identified multiple uterine myomas occupying the pelvis and the left ovary, with oedematous swelling that had moved ventrally to the uterus. She was diagnosed with adnexal torsion and underwent emergency laparoscopic adnexectomy. Nine months after surgery, she experienced right lower abdominal pain. Ultrasonography revealed suspected right adnexal torsion and she underwent emergency surgery. The right ovary was twisted 540° counterclockwise and swollen to 7 cm in size, with partial polycystic changes. She was histopathologically diagnosed with a PCO, and the final diagnosis, which also considered the endocrine test results, was PCOS. In PCOS, adnexal torsion may occur if the swollen ovary moves because of a hysteromyoma or other cause. Accordingly, torsion should be considered during the follow-up of patients with PCOS.

Outcome of Neoadjuvant Intra-Arterial Chemotherapy and Radical Hysterectomy for Treatment of Bulky Stage IB to Stage IIB Uterine Cervical Cancer: Can Postoperative Irradiation Be Avoided?

Objectives We evaluated whether our neoadjuvant intra-arterial chemotherapy (NAIC) effectively precludes the need for postoperative radiation therapy in patients treated by radical hysterectomy for IB2 to IIB cervical cancer. Materials and Methods Study subjects were 52 patients with a bulky cervical tumor diagnosed and treated at Juntendo University Hospital or Juntendo Nerima Hospital. The NAIC combined cisplatin, epirubicin, mitomycin-C, and 5-fluorouracil; and radical hysterectomy was to be performed after 2 cycles. The main variables analyzed were clinical and histologic response to NAIC, NAIC-related adverse events, adjuvant chemotherapies, relapse-free and overall survival, recurrence, and prognostic factors. Results Patients were judged eligible for radical hysterectomy, and 51 underwent the surgery. The overall positive response (complete response [CR] + partial response [PR]) to NAIC was 88.5%. Median follow-up time was 84 months (5–136 months). Three-year relapse-free survival and overall survival were 80.5% and 77.8%, respectively. The recurrence rate was 19.2% (10/52 patients). Seven (13.5%) of the 52 patients died from the disease during follow-up. Lymph node status (positive vs negative) and the histologic effect of NAIC (grades 0–1 vs grades 2–3) were shown to be prognostic factors (P = 0.024 and P = 0.021, respectively). Conclusions Our NAIC strategy seems to be well tolerated and beneficial for patients with bulky IB2 to IIB cervical cancer. With this strategy, radiation therapy remains an option in cases of recurrence. For cases in which lymph node metastasis is found or the histologic effect of NAIC is low, our adjuvant chemotherapy regimen may need adjustment to improve prognosis.

Side-population Cells Derived from Non-tumorigenic Rat Endometrial Cells are a Candidate Cell of Origin for Malignant Endometrial Tumors

We have previously demonstrated that side population (SP) cells derived from endometrial cancer cells have cancer stem-like cell features. However, the role of stem cell-enriched subpopulations, SP cells in normal endometrium for carcinogenesis is still poorly understood. In the present study, to modeled early carcinogenesis in normal endometrium, we established two cell lines by introducing the oncogenic KRAS gene into SP (RSP) cells and non-SP (RNSP) cells from a rat non-tumorigenic endometrial cell line. Tumorigenicity was enhanced in SP cells harboring mutant KRAS (RSP-K12V cells) compared with that in NSP harboring mutant KRAS gene (RNSP-K12V cells). The primary cultured tumor cells derived from RSP-K12V cells exhibited long-term proliferating capacity in culture and had the capacity to form serial tumors in vivo. In contrast, the primary cultured tumor cells derived from RNSP-K12V cells failed to grow and became senescent. The proportion of SP cells was higher in RSP-K12V cells than in RSP cells and was highest in the RSP-K12V tumor cells and it was correlated with tumorigenicity. The levels of c-Myc and Oct4, and the transcriptional activity of the estrogen receptor were enhanced in RSP-K12V cells and their tumor cells compared with those in RNSP-K12V cells and their tumor cells, respectively. Tumor cells derived RSP-K12V acquired the potential for estrogen-independent proliferation. This is the first report which demonstrates that the occurrence of KRAS gene mutations in SP cells rather than NSP cells derived from nontumorigenic endometrial cells, contributes to the development of malignant endometrial tumors.