Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis

Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective.     

Effect of sevoflurane on Ca2+ mobilization in Madin-Darby canine kidney cells

We investigated the effect of the volatile anesthetic sevoflurane on Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells by using the fluorescent dye fura-2/AM (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaace-toxymethyl ester) as the Ca2+ indicator. At a concentration of 0.15 mM, sevoflurane did not alter basal cytosolic free calcium concentration ([Ca2+]i); however, at concentrations of 0.45-0.6 mM, sevoflurane did elevate [Ca2+]i, mainly by releasing Ca2+ from the endoplasmic reticulum (ER) store. Sevoflurane (0.15 mM) did not change either the [Ca2+]i peak evoked by high doses of ATP or UTP or inhibition of the ER Ca2+ pump, although it did significantly slow down the decay of the [Ca2+]i rise. Lastly, sevoflurane inhibited the capacitative Ca2+ entry and Mn2+ quench of fura-2 fluorescence induced by Ca(2+)-mobilizing ligands.     

Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

Oral administration of OK-432 (picibanil). (8th report) clinical application: its immunomodulatory effects on the patients with gastrointestinal cancers

A streptococcal preparation, OK-432 at a dose of 5 KE was orally administered to the patients with gastric or colorectal cancer for 7 approximately 14 days before operation, and its immunomodulatory effects on peripheral blood lymphocytes (PBL), regional lymph node lymphocytes (RLNL) and tumor infiltrating lymphocytes (TIL) were assessed. OK-432 treated group included 5 gastric and 6 colorectal cancers, and control group included 6 gastric and 8 colorectal cancers. After oral administration of OK-432 the proportion of Leu 7+ and Leu 11+ cells in PBL increased, and NK cell activity of PBL also was augmented. The proportion of OKT8+ cells increased in PBL and those of OKT3+ cells and OKT8+ cells decreased in RLNL after oral administration of OK-432. The responsiveness of TIL to autologous tumor extracts in the presence of interleukin-2 was enhanced in oral OK-432 group. These results indicate that oral OK-432 affects on NK and T cells and augments the antitumor immunity of the patients with gastrointestinal cancer.     

Postoperative low-pelvic irradiation for stage I-IIA cervical cancer patients with risk factors other than pelvic lymph node metastasis

: To retrospectively investigate whether postoperative low-pelvic radiotherapy (RT) is an appropriate treatment for node-negative, high-risk Stage I–IIA cervical cancer patients.

: A total of 228 Stage I–IIA cervical cancer patients treated by radical surgery and postoperative RT were included in this study. All patients had histopathologically negative pelvic node metastasis, but at least one of the following risk factors: parametrial involvement, positive or close resection margins, invasion depth two-thirds or greater cervical stromal thickness. Seventy-nine patients (35%) received 30–50 Gy (median 44) to whole pelvis and a boost dose to the low pelvis (whole-pelvic RT group); the other 149 patients (65%) received low-pelvic RT only (low-pelvic RT group). For both groups, the total external RT dose to the low pelvis ranged from 40 to 60 Gy (median 50). The potential factors associated with survival, small bowel (gastrointestinal) complications, and leg lymphedema were analyzed, and patients who had a relapse in the upper pelvis were identified.

: The 5-year overall and disease-specific survival rate was 84% and 86%, respectively. After multivariate analysis, only bulky tumor (≥4 cm) and non-squamous cell carcinoma were significantly associated with survival. Parametrial involvement, lymph-vascular invasion, ≤50.4 Gy to the low pelvis, positive or close margins, and low-pelvic RT alone did not significantly affect survival. Grade I–V small bowel complications occurred in 33 patients (15%). Whole pelvic RT and >50.4 Gy to the low pelvis, but not old age and treatment technique (AP-PA vs. box), were significantly associated with gastrointestinal complications. Three patients (2%) in the low-pelvic RT group and 6 patients (8%) in the whole-pelvic RT group were found to have Grade III or higher small bowel complications (p = 0.023). Thirty-one percent of patients developed lymphedema of the leg. A dose to the low pelvis >50.4 Gy and an AP-PA field, but not whole-pelvic RT, old age, or the number of sampled lymph nodes, were associated with lymphedema of the leg. Five patients (3.6%) of the low-pelvic RT group and none of the whole-pelvic RT group developed upper pelvis relapse. Three of these 5 patients had upper pelvic relapse alone.

: Compared with whole-pelvic RT plus low-pelvic boost, low-pelvic RT alone significantly reduces the small bowel complications in node-negative, high-risk, Stage I–IIA cervical cancer patients. Although low-pelvic RT alone increases the incidence of upper pelvic relapse, its effect on survival is not substantial. Low-pelvic RT alone appears to be an appropriate treatment method for this group of patients.     

Quantitative Analysis of Multidrug-resistance mdr1 Gene Expression in Head and Neck Cancer by Real-time RT-PCR

Progression of head and neck cancer is always associated with changes of gene expression profile. In this study, we characterized the expression of multidrug‐resistance mdr1 gene, which may play a role in tumorigenesis and multidrug resistance in head and neck cancer. A TaqMan one‐step RT‐PCR with a linear range for quantification across at least a 5 log scale of concentration of mdr1 mRNA was designed to determine the level of mdr1 expression in 50 pairs of normal vs. malignant head and neck tissues. Both the absolute level of mdr1 mRNA in tumor (T) and the relative mdr1 expression between tumor and its normal counterpart (T/N) were measured and their associations with several clinical variables were analyzed. Among the clinical variables analyzed, only the clinical stage of tumor was found to be associated with mdr1 expression. The distribution of clinical stages differed significantly (P<0.01) among the 27 specimens that had a T/N>1, with 59.3%, 22.2%, 14.8% and 3.7% in stage IV, III, H, and I, respectively. In addition, 76% of stage IV and 75% of stage III tumors had a T/N>1 compared to 25% of stage II and 20% of stage I tumors (P=0.004). Multivariate logistic regression analysis also indicated a significant difference of mdr1 expression between the early (I and II) and advanced (III and IV) stages tumors. The adjusted odds ratios (95% confidence intervals) were 1.477 (1.084–2.012) and 1.001 (1.000–1.002) for T/N (P<0.05) and T (P<0.05) treated as continuous variables, and 15.521 (3.414–70.550) and 5.074 (1.154–22.311) for T/N (P<0.001) and T (P<0.05) treated as binary variables, respectively. Taken together, the data presented here indicated that real‐time RT‐PCR provides a quantitative way to monitor mdr1 gene expression. The differential expression of mdr1 between early and advanced stages of head and neck cancer may shed light on the process of tumorigenicity and offer clues to the planning of new treatments.     

Inhibition of cell cycle progression in human leukemia HL-60 cells by esculetin

Esculetin, a coumarin compound, was found to inhibit cell growth and cell cycle progression by inducing arrest of the G1 phase in HL-60 cells. To obtain information regarding cell cycle arrest induced by esculetin, we examined its effect on the regulating factors of the G1 phase in the leukemia HL-60 cells treated with esculetin by Western blotting. Our observations were: (1) a distinct increase in the level of hypophosphorylated retinoblastoma protein (pRb), and a reduction in the level of CDK4 after treatment with 100 microM of esculetin for 24 h; (2) a marked up-regulation of p27, and a down-regulation of cyclin D1 after treatment with 100 microM esculetin for 24 h. These results suggest that esculetin can inhibit the growth of human leukemia HL-60 cells by G1 phase cell cycle arrest as a result of inhibited pRb phosphorylation.     

Tumor-specific promoter-driven adenoviral therapy for insulinoma

Background

Insulinomas are the most common type of neuroendocrine (NE) pancreatic islet tumors. Patients with insulinomas may develop complications associated with hyperinsulinemia. To increase the treatment options for insulinoma patients, we have tested a conditionally replicating adenovirus that has been engineered in such a way that it can specifically express therapeutic genes in NE tumors.

Methods

We used a promoter-specific adenoviral vector delivery system that is regulated by an INSM1 (insulinoma-associated-1) promoter, which is silent in normal adult tissues but active in developing NE cells and tumors. Through a series of modifications, using an insulator (HS4) and neuron-restrictive silencer elements (NRSEs), an oncolytic adenoviral vector was generated that retains tumor specificity and drives the expression of a mutated adenovirus E1A gene (Δ24E1A) and the herpes simplex virus thymidine kinase (HSV-tk) gene. The efficacy of this vector was tested in insulinoma-derived MIN, RIN, βTC-1 and pancreatic (Panc-1) cells using in vitro cell survival and in vivo tumor growth assays.

Results

Using in vitro insulinoma-derived cell lines and an in vivo subcutaneous mouse tumor model we found that the INSM1 promoter-driven viruses were able to replicate specifically in INSM1-positive cells. INSM1-specific HSV-tk expression in combination with ganciclovir treatment resulted in dose-dependent tumor cell killing, leaving INSM1-negative cells unharmed. When we combined the INSM1-promoter driven HSV-tk with Δ24E1A and INSM1p-HSV-tk (K5) viruses, we found that the co-infected insulinoma-derived cells expressed higher levels of HSV-tk and exhibited more efficient tumor suppression than cells infected with INSM1p-HSV-tk virus alone.

Conclusions

INSM1 promoter-driven conditionally replicating adenoviruses may serve as a new tool for the treatment of insulinoma and may provide clinicians with additional options to combat this disease.

     

Management of infectious scleritis after pterygium excision

PURPOSE: We sought to describe the clinical features, responsible pathogens, management, and prognosis of infectious scleritis after pterygium excision. METHODS: A retrospective study through review of medical records of patients diagnosed with infectious scleritis after pterygium excision over a 10-year period at our institution. RESULTS: A total of 16 cases of infectious scleritis after pterygium excision was identified. Among them, eight were associated with sclerokeratitis, and six had multifocal scleral nodules with subconjunctival abscesses. Culture results were positive in 15 (93.8%) cases. Pseudomonas was isolated in 13 (81.3%) patients, fungus in three (18.8%), and two had a mixed growth (12.5%). Based on the in vitro susceptibility test, four (31%) Pseudomonas isolates were resistant to gentamicin, whereas all isolates were sensitive to amikacin. During the course of treatment, eight cases were complicated by vitreous opacity, four developed glaucoma, four had serous retinal or choroidal detachment, and two had secondary cataract. Scleral infection recurred in two patients after cessation of therapy. Among the nine patients treated with medical therapy, two eyes were enucleated, whereas only two attained a visual acuity of > or =2/200 at the end of the follow-up period. On the other hand, seven patients had combined antibiotic therapy and surgical debridement. The number of surgical debridement ranged from one to three, with an average of 1.4. In this combined-treatment group, only one patient required enucleation, and five cases attained a visual acuity of > or =2/200. The duration of hospitalization for patients with combined treatment was 21.2+/-4.8 days compared with the 28.4+/-5.0 days for those with medical treatment alone (p = 0.035). CONCLUSION: Surgical debridement in combination with appropriate antimicrobial therapy shortens the course of treatment and improves the visual outcome of severe infectious scleritis after pterygium excision.