Comprehensive assessment of the prognosis of pancreatic cancer: peripheral blood neutrophil-lymphocyte ratio and immunohistochemical analyses of the tumour site.

OBJECTIVE: Several studies have suggested that an elevated neutrophil-lymphocyte ratio (NLR) is associated with a poorer prognosis in patients with pancreatic cancer (PC). The correlations between the NLR and immunohistochemical (IHC) analysis with regard to the prognosis of patients with PC remain to be elucidated. By using IHC findings, we determined the value of the NLR as a prognostic factor in patients with PC. MATERIAL AND METHODS: We collected the clinico-pathological data of 28 consecutive patients who underwent surgical resection for PC between January 2008 and December 2012 at The Jikei University Kashiwa Hospital. We investigated whether the NLR and IHC results were related and ensured the consistency of the prognosis of patients with PC. RESULTS: The Kaplan-Meier curves for the disease-free survival (DFS) and the overall survival (OS) revealed that an NLR ≥ 5 is an implicit factor for decreased DFS and OS in patients with PC (p = 0.003, p < 0.001, log-rank test). The density of CD163(+) macrophages and CD66b(+) neutrophils was significantly higher in the high NLR group; on the contrary, the density of CD20(+) lymphocytes was significantly higher in the low NLR group. Moreover, a Mann-Whitney U test showed that the NLR was significantly correlated with a high density of CD20(+) lymphocytes (p = 0.031) and CD163(+) macrophages (p = 0.023), while the NLR was not significantly correlated with CD66b(+) neutrophils (p = 0.397). CONCLUSIONS: Our results demonstrated the validity of the NLR by IHC analyses and we determined that a higher value of NLR is a trustworthy prognostic factor for patients with PC.

Targeting Id1 reduces proliferation and invasion in aggressive human salivary gland cancer cells.

BACKGROUND: Salivary gland cancer (SGC) is one of the common malignancies of the head and neck area. It develops in the minor and major salivary glands and sometimes metastasizes to other organs, particularly to the lungs. Inhibitors of differentiation (Id) proteins are negative regulators of basic helix-loop-helix transcription factors that control malignant cell behavior and tumor aggressiveness in many tissues. In this study, our goal was to determine the potential role of Id proteins, particularly Id1, during human SGC cell progression. METHODS: We first determined the expression levels of Id1 and Id2 in four SGC cell lines: two adenocarcinoma of the salivary gland (HSG and HSY) and two adenoid cystic carcinoma (ACC2 and ACCM) cell lines. We then used constructs that expressed antisense cDNAs to Id1 or Id2 to knockdown the expression of these proteins in cell lines where they were highly expressed, and determined the effects of the knockdown on cell proliferation, migration and invasion. RESULTS: Id1 mRNA and protein were detectable in all cell lines, and expression of Id2 was variable, from absent to high. The ACC2 and ACCM cell lines expressed both Id1 and Id2, but Id1 was expressed at a higher level in the more aggressive ACCM cell line in comparison to ACC2 cells as confirmed by Id1 promoter-reporter assays. We therefore focused on the ACCM cells for the remainder of the study. We found that proliferation and invasiveness of ACCM cells were strongly reduced after Id1 knockdown whereas Id2 suppression had only a slight effect. Results of scratch and colony formation assays also confirmed that ACCM cell aggressiveness was significantly reduced upon Id1 knockdown. Finally, this knockdown resulted in reduced c-myc and enhanced cyclin-dependent kinase inhibitor p21 expression. CONCLUSIONS: These results demonstrate that Id1 plays an important role in the control of human SGC cell aggressiveness and suggest a potential role as a marker of diagnosis, prognosis and progression of SGCs. Id1 suppression could represent a novel and effective approach for the treatment of salivary gland cancer.

Silencing Id-1 with RNA interference inhibits adenoid cystic carcinoma in mice.

BACKGROUND: The helix-loop-helix (HLH) protein Id-1 (inhibitor of DNA binding/differentiation) has been demonstrated to play an important role in tumor development. Our previous in vitro research has shown that Id-1 is a potential target in the treatment of human adenoid cystic carcinoma (ACCM). The purpose of this study was to analyze the influence of Id inhibition on ACCM in mice. MATERIALS AND METHODS: To suppress the expression of Id-1 gene, we used lentivirus-mediated RNA interference to silence the Id-1 gene post-transcriptionally in ACCM models that stably express GFP in mice. Tumor development was evaluated by size measurement. Effects of Id-1 siRNA on mRNA and protein expression of Id-1 were analyzed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting respectively. Ki-67 expression was measured by immunohistochemistry. In vitro studies of Hoechst staining for cell apoptosis, Boyden-chamber assay for cell invasion, and MTT-tests for cell growth were performed as well. RESULTS: Id-1 knockdown resulted in inhibition of tumor growth in mice. Id-1 siRNA significantly decreased not only Id-1 in mRNA and protein level, but also Ki-67 expression. In addition, apoptosis was induced and cell proliferation activity and invasion were significantly reduced. CONCLUSIONS: Lentivirus-mediated gene knockdown by silencing Id-1 constitute a valid methodological approach, which may represent an attractive, potent and specific therapeutic tool for the treatment of ACCM.

Carrier cell-mediated cell lysis of squamous cell carcinoma cells by squamous cell carcinoma antigen 1 promoter-driven oncolytic adenovirus.

BACKGROUND: The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes: SCCA1 and SCCA2. SCCA1 gene is up-regulated in squamous cell carcinoma cells. We analyzed the proximal region of the SCCA1 promoter and the antitumor effect of oncolytic adenovirus driven by the SCCA1 promoter in squamous cell carcinoma cells. METHODS: The SCCA1 promoter was analyzed by dual luciferase assay and substituted with the E1A promoter to construct the oncolytic adenovirus to determine the squamous cell carcinoma-specific cell lysis. RESULTS: Deletion analysis of SCCA1 promoter identified a 175-bp core promoter region and an enhancer region at -525 to -475 bp upstream of the transcription start site. The transcriptional activity of the SCCA1 promoter was up-regulated in squamous cell carcinoma cells. Five tandem repeats of enhancer increased SCCA1 promoter activity by four-fold. Oncolytic adenovirus driven by this SCCA1 enhancer-promoter complex specifically killed squamous cell carcinoma cells in vitro and in vivo. A549 carrier cells infected with the oncolytic adenovirus induced complete regression of syngeneic squamous cell carcinoma cell tumor by overcoming immunogenicity and adenovirus-mGM-CSF augmented the antitumor effect of carrier cells. CONCLUSIONS: SCCA1 was up-regulated in squamous cell carcinoma cells and oncolytic adenovirus driven by SCCA1 promoter specifically killed these cells. These findings suggest that SCCA1 promoter is a potential target of gene therapy for squamous cell carcinoma.

Anti-tumor effect of small interfering RNA targeting the androgen receptor in human androgen-independent prostate cancer cells.

Early phase prostate cancer is usually androgen-dependent, with the androgen/androgen receptor (AR) signaling pathway playing a central role. At this stage, the cancer responds well to androgen ablation therapy, but prostate cancers eventually acquire androgen independence and more aggressive phenotypes. Several studies, however, have shown that the majority of tumors still express functional AR, which is often amplified and mutated. To determine if the AR is a plausible therapeutic target, we investigated the anti-tumor effect of small interfering RNAs targeting the AR (siAR) in the human prostate cancer cells, LNCaP and 22Rv1, which express mutated AR. In both types of cells, transfection of siAR suppressed mutated AR expression and significantly reduced cell growth. Furthermore, atelocollagen-mediated systemic siAR administration markedly inhibited the growth of 22Rv1 cells subcutaneously xenografted in castrated nude mice. These results suggest that the AR is still a key therapeutic target even in androgen-independent prostate cancer (AIPC). Silencing of AR expression in AIPC opens promising therapeutic perspectives.

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo.

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.

Massive subcutaneous emphysema developing before surgery for mandibular fracture: a case report.

Preoperative massive subcutaneous emphysema before intubation is extremely rare. However, this complication may be potentially lethal, depend on the condition of air spreading. Subcutaneous emphysema which occurs intra- or postoperative period is sometimes iatrogenic because the air is introduced into the tissue space through the hole injured by the operation. But the emphysema in this case occurred preoperatively by the pressure of the bag valve mask, because the patient had an intra-oral wound, which reaches the submental space. In this report, we describe an extremely rare case of preoperative massive emphysema of the patient with the mandibular fracture.

CD151 regulates HGF-stimulated morphogenesis of human breast cancer cells.

We previously demonstrated that CD151 forms a functional complex with c-Met and integrin alpha3/alpha6 in human salivary gland cancer cells. In the current study, we investigated the involvement of CD151, c-Met, and integrin alpha3/alpha6 in the cellular morphogenesis of human breast cancer cells. Knockdown of CD151, integrin alpha3, or integrin alpha6 expression abolished branching morphogenesis. Decreased c-Met expression in these cells led to the formation of rudimentary networks and prevented their conversion. Furthermore, hepatocyte growth factor (HGF) promoted cellular morphogenesis by accelerating network reorganization. Immunoprecipitation revealed a specific association between CD151 and c-Met. The involvement of CD151 and integrin alpha3/alpha6 in HGF-dependent signaling was confirmed by the decreased Akt phosphorylation in cells lacking CD151, integrin alpha3, or integrin alpha6. Hence, the regulation of CD151 expression might contribute to changes in HGF/c-Met signaling and thereby modulate the phenotypic characteristics of cancer cells.

Hypoxia enhances CXCR4 expression by activating HIF-1 in oral squamous cell carcinoma.

Hypoxia promotes the invasive and metastatic potential of tumour cells. A recent study has shown that the activation of the chemokine receptor CXCR4 by lack of oxygen in breast cancer is HIF-1-dependent. We have previously demonstrated that CXCR4 signalling is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (OSCC). In this study, we investigated a correlation between CXCR4 and HIF-1alpha expression in OSCC. Immunohistochemistry showed that CXCR4 was expressed in 20 of 85 OSCC tissues, while HIF-1alpha was expressed in 51 of 85 samples. There was a significant correlation between the expression of CXCR4 and HIF-1alpha. In human OSCC cells, hypoxia markedly enhanced the expression of both HIF-1alpha and CXCR4. Furthermore, synthetic small interfering RNA specific for HIF-1alpha significantly suppressed the expression of this protein, and also attenuated the induction of CXCR4 expression under hypoxic conditions. These results indicated that HIF-1alpha regulated hypoxia-induced CXCR4 expression in OSCC.

Role of Akt isoforms in HGF-induced invasive growth of human salivary gland cancer cells.

The expression of hepatocyte growth factor (HGF) and c-Met is associated with tumor progression in many human malignancies. A recent study demonstrated HGF and c-Met expression in human salivary gland cancer tissues. Here, we investigated the role of the HGF/c-Met system in the invasive growth of two human salivary gland cancer cell lines: green fluorescent protein-adenoid cystic carcinoma 2 (GFP-ACC2) and GFP-ACCM. HGF enhanced the invasive growth of the two cell lines by activating PI3K/Akt signaling. All Akt isoforms (Akt1, Akt2, and Akt3) were detected in both cell types by Western blot analysis. Knockdown of any of the Akt isoforms using isoform-specific synthetic small-interfering RNAs largely abrogated the invasive growth induced by HGF. Our findings suggest that all of the Akt isoforms are required for the HGF-stimulated invasive growth of human salivary gland cancer cells, and that targeting a single Akt isoform could be effective in treating salivary gland cancers.

Stat3 as a molecular target in RNA interference-based treatment of oral squamous cell carcinoma.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been observed in many human malignancies. Using the sequence-specific RNA interference (RNAi) method to switch off Stat3 expression, it may be possible to arrest cancer growth. In this study, we aimed to identify the most effective sequence of a synthetic small interfering RNA (siRNA) specific for Stat3 (Stat3-siRNA) and the effect of Stat3 suppression on the growth of oral squamous cell carcinoma cells. Ten designed siRNAs with known sequences were screened for the best RNAi effect at the working concentrations of 1 and 10 nM. The range of reduction of Stat3 expression varied from 21 to 67% for 10 nM siRNAs, and from 13 to 73% for 1 nM siRNAs. Three out of the 10 screened siRNAs reduced Stat3 expression to lower levels compared with the GFP-siRNA control. The interferon response of some siRNAs was observed at a concentration of 10 nM. However, at 1 nM, the mRNA levels of interferon response genes (OAS1, OAS2, MX1 and ISFG3gamma) remained unchanged. The growth of GFP-SAS, HSC-3, HSC-4 and KB cells was strongly inhibited by the use of three effective Stat3-siRNAs in comparison with other Stat3-siRNAs and GFP-siRNA. Moreover, the mRNA levels of genes for which transcription is activated by Stat3 were markedly suppressed. These results suggest that targeting Stat3 using siRNA may constitute a useful approach for the treatment of oral squamous cell carcinoma.

Multicenter retrospective study of cetuximab plus platinum-based chemotherapy for recurrent or metastatic oral squamous cell carcinoma.

PURPOSE: The purpose of this study was to assess the efficacy and safety of cetuximab plus platinum-based chemotherapy for patients specifically diagnosed with recurrent or metastatic oral squamous cell carcinoma (OSCC). METHODS: We conducted a multicenter retrospective observational study of patients who underwent first-line cetuximab plus platinum-based chemotherapy between December 2012 and June 2015. 65 patients received weekly cetuximab (week 1, 400 mg/m2; subsequent weeks, 250 mg/m2) plus a maximum of six 3-weekly cycles of cisplatin (80 or 100 mg/m2, day 1) or carboplatin (at an area under the curve of 5 mg/mL/min as a 1-h intravenous infusion on day 1) and 5-fluorouracil (800 or 1000 mg/m2/day, days 1-4). Patients with stable disease who received cetuximab plus platinum-based chemotherapy continued to receive cetuximab until disease progression or unacceptable toxicities, whichever occurred first. RESULTS: The median follow-up was 10.5 (range 1.2-34.2) months. The best overall response and the disease control rates were 46.2 and 67.7%, respectively. The median overall survival and progression-free survival rates were 12.1 and 7.8 months, respectively. The most common grades 3-4 adverse events were skin rash (9.2%) followed by leukopenia (6.2%). None of the adverse events were fatal. CONCLUSION: The results of our multicenter retrospective study, which was the largest of its kind to date, suggest that first-line cetuximab plus platinum-based chemotherapy is suitable and well-tolerated for the systemic therapy of recurrent or metastatic OSCC.

Prognostic impact of preoperative serum interleukin-6 levels in patients with early-stage oral squamous cell carcinoma, defined by sentinel node biopsy.

Failure to detect recurrence and lymph node metastasis early represents a fundamental barrier to the improvement of survival rate in early stage oral squamous cell carcinoma (OSCC). The present study evaluated the association between serum interleukin-6 (IL-6) level and clinical outcomes in patients with early stage OSCC patients defined by sentinel node biopsy (SNB). A total of 53 patients with clinical stage I/II OSCC who underwent SNB were enrolled. SNB was determined by a radioisotope method, and was evaluated by histopathological examination and genetic analysis. Preoperative sera were measured for IL-6 by ELISA. In the clinical stage I/II patients, disease-free survival (DFS) was demonstrated to be higher in patients with negative SNB compared with patients with positive SNB. In total, 13 patients were demonstrated to exhibit lymph node metastasis by SNB or were reclassified to pathological stage T4 subsequent to analysis of the surgically resected specimens. Thus, 40 patients were diagnosed with early stage OSCC. Of these 40 patients, DFS of the patients with low serum IL-6 was significantly higher compared with the patients with high serum IL-6 (P=0.012). In 19 patients with negative SNB and low serum IL-6, the disease-free rate was 100%. These findings suggested that SNB staging and serum IL-6 level have a high prognostic value in patients with early stage OSCC. Additional investigation and longer follow-up times are warranted to improve understanding of the group of patients that may benefit from this procedure.

The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

Palonosetron Prevents Highly Emetogenic Chemotherapy-induced Nausea and Vomiting in Oral Cancer Patients.

BACKGROUND/AIM: To evaluate the efficacy of palonosetron in preventing acute and delayed nausea and vomiting in patients receiving highly emetogenic chemotherapy (HEC) in oral cancer patients. PATIENTS AND METHODS: Oral cancer patients receiving HEC were enrolled; among the 40 patients, 87 courses of chemotherapy were administered. On day 1, 0.75 mg palonosetron was intravenously administrated just before chemotherapy. RESULTS: The primary endpoint was the proportion of patients with a complete response (CR) and the secondary endpoint was the proportion of patients with complete control (CC) during the acute and delayed phase. During the acute phase, 86 of 87 courses (98.9%) had CR and 84 of 87 courses (96.6%) had CC. During the delayed phase, 84 of 87 courses (96.6%) had CR and 70 of 87 courses (80.5%) had CC. CONCLUSION: Palonosetron is effective at preventing HEC-induced chemotherapy-induced nausea and vomiting (CINV) in oral cancer chemotherapeutic regimens in the acute and delayed phases.

Corrigendum: The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells.

This corrects the article DOI: 10.1038/srep42845.

CXCR4 expression is associated with lymph-node metastasis of oral squamous cell carcinoma.

The support mechanisms that are involved in lymph-node metastasis of oral squamous cell carcinoma (OSCC) remain largely unknown. Recent studies have demonstrated that tumor cells express chemokine receptors and use chemokines to metastasize to the target organ in many malignancies in humans. In this study, we examined the expression and function of chemokines and their receptors in OSCC. The expression of chemokine receptors was assessed in eight OSCC cell lines. CXCR3 mRNA and protein were expressed in all the OSCC cell lines examined, while CXCR4 mRNA and protein were expressed only in HSC2, HSC3, and Ca9-22 cells. Treatment with the ligand for CXCR4, stromal cell-derived factor-1 (SDF-1), enhanced the motility and invasiveness of OSCC cells expressing CXCR4. However, the CXCR3 ligand, Mig, did not affect the migration or invasiveness of CXCR3-positive cells. We also evaluated the clinical significance of CXCR4 expression immunohistochemically. CXCR4 expression was detected in 27 (30%) of the 90 OSCC tissues tested, and was localized in the membrane and cytoplasm of cancer cells. There was a highly significant correlation between CXCR4 expression and lymph-node metastasis (P=0.0035). Collectively, these findings suggest that CXCR4 might be involved in the lymph-node metastasis of OSCC.

Anti-tumor effect of radiation response by combined treatment with angiogenesis inhibitor, TNP-470, in oral squamous cell carcinoma.

Blocking angiogenesis may enhance conventional anticancer treatments such as radiation therapy. In this study, we examined the effects of the angiogenesis inhibitor TNP-470 on human OSCC cell lines HSC2 and KB, with combining radiation therapy in the nude mouse. We evaluated cell-induced neovascularization with dorsal air sac assay, and selected two cells (HSC2: low, KB: high) with different level of cell-induced angiogenesis. The angiogenesis inhibitor TNP-470 was given 30 mg/kg s.c. daily on day 1-5, and irradiation, 8 Gy x 1, was administered on day 1 each week for 3 weeks. Significant inhibition of tumor growth relative to untreated controls was achieved in KB cells showing high induced angiogenesis with both TNP-470 (P < 0.01) and radiation (P < 0.01) and combining TNP-470 and radiation (P < 0.01). We saw little effect of TNP-470 either alone or in addition to the effect of radiation on the HSC2 cells showing low induced angiogenesis. These results suggested that TNP-470 significantly enhanced the effect of radiation on the cells with high neovascularization. These findings indicated that individual evaluation of each tumor neovascularization potential will be important before deciding the anti-angiogenesis treatment.

Hypoxia enhances c-Met/HGF receptor expression and signaling by activating HIF-1alpha in human salivary gland cancer cells.

Hypoxia increases the invasive and metastatic potential of tumor cells. Increased expression of c-Met/hepatocyte growth factor (HGF)-receptor protein in response to hypoxia in thyroid papillary carcinomas is hypoxia-inducible factor-1 (HIF-1) dependent. Both HGF and c-Met are expressed in human salivary gland cancers. In the current study, we tested whether c-Met expression was regulated by hypoxia and HIF-1alpha using two human salivary gland cancer cell lines: GFP-ACC2 and GFP-ACCM. Hypoxia enhanced the expression of HIF-1alpha in both cell lines, whereas c-Met was markedly induced only in the GFP-ACCM cells, which have metastatic potential. In the latter, hypoxia also promoted HGF-induced invasiveness. Synthetic small-interfering RNA specific for HIF-1alpha inhibited HIF-1alpha expression in the GFP-ACCM cells, and also suppressed the increase in c-Met expression and HGF-induced invasiveness under hypoxic conditions. These results suggest that hypoxia activates the HGF/c-Met system via HIF-1alpha in human salivary gland cancers and might be involved in their metastasis.

Reduction of human metastatic breast cancer cell aggressiveness on introduction of either form a or B of the progesterone receptor and then treatment with progestins.

The sex steroid hormone progesterone (Pg) is critically involved in the development of the mammary gland, and it also is thought to play a role in breast cancer progression. However, the effect of Pg on malignant phenotypes is not fully understood in breast cancer. We previously reported that in Pg receptor (PR)-positive T47D breast cancer cells, Pg was able to counterbalance the stimulatory effect of estrogen or serum on proliferation and on expression level of Id-1, which generally stimulates cell proliferation and inhibits differentiation. Conversely, metastatic MDA-MB231 breast cancer cells lack PR and express high levels of Id-1 constitutively, and Pg showed no effect on Id expression, proliferation, and invasion in these cells. However, after introducing PR (either PR-A or PR-B) into MDA-MB231 cells, Pg inhibited the expression of Id-1 mRNA drastically. PR-transfected MDA-MB231 cells exhibited less proliferative activity after Pg treatment than parental or control MDA-MB231 cells, an effect which correlated well with reduction of Id-1 mRNA. This inhibitory effect on proliferation was accompanied by p21 up-regulation and c-myc down-regulation. Moreover, Pg-treated PR transfectants showed significant morphologic change, appearing more flattened and spread out than control ethanol-treated cells. Boyden chamber invasion assay revealed that PR-transfected MDA-MB231 cells also lost most of their invasive properties after Pg treatment. Zymographic analysis revealed that Pg drastically inhibited matrix metalloproteinase-9 (MMP-9) activity in cells transfected with either PR-A or PR-B. To determine whether Id-1 could act as a key mediator of the effects of Pg, we prepared cells transfected with Id-1 and PR. The morphologic change and p21 up-regulation still were observed after Pg treatment. However, c-myc down-regulation was not observed; the proliferative and invasive activities were mostly recovered; and MMP-9 down-regulation could not be detected anymore. From these observations, we conclude that either form of the PR is sufficient to reduce the malignant phenotypes on treatment with Pg and that Id-1 plays an important role as a mediator of the effects of Pg on breast cancer cell proliferation and invasion.