Cholesterol sulfate inhibits osteoclast differentiation and survival by regulating the AMPK-Sirt1-NF-κB pathway.

Cholesterol sulfate (CS) is an activator of retinoic acid-related orphan receptor α (RORα). CS treatment or RORα overexpression attenuates osteoclastogenesis in a collagen-induced arthritis mouse model. However, the mechanism by which CS and RORα regulate osteoclast differentiation remains largely unknown. Thus, we aimed to investigate the role of CS and RORα in osteoclastogenesis and their underlying mechanism. CS inhibited osteoclast differentiation, but RORα deficiency did not affect osteoclast differentiation and CS-mediated inhibition of osteoclastogenesis. CS enhanced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) activity, leading to nuclear factor-κB (NF-κB) inhibition by decreasing acetylation at Lys310 of p65. The NF-κB inhibition was restored by AMPK inhibitor, but the effects of CS on AMPK and NF-κB were not altered by RORα deficiency. CS also induced osteoclast apoptosis, which may be due to sustained AMPK activation and consequent NF-κB inhibition, and the effects of CS were significantly reversed by interleukin-1ß treatment. Collectively, these results indicate that CS inhibits osteoclast differentiation and survival by suppressing NF-κB via the AMPK-Sirt1 axis in a RORα-independent manner. Furthermore, CS protects against bone destruction in lipopolysaccharide- and ovariectomy-mediated bone loss mouse models, suggesting that CS is a useful therapeutic candidate for treating inflammation-induced bone diseases and postmenopausal osteoporosis.

ACAT-1-Regulated Cholesteryl Ester Accumulation Modulates Gemcitabine Resistance in Biliary Tract Cancer.

BACKGROUND: Biliary tract cancer (BTC) has few choices of chemotherapy, including gemcitabine, therefore exploring the mechanisms of gemcitabine resistance is important. We focused on lipid metabolism because biliary tract epithelial cells are essential in cholesterol and bile acid metabolism and the messenger RNA (mRNA) microarray analysis showed high acyl coenzyme A: cholesterol acyltransferase 1 (ACAT-1) expression in BTC gemcitabine-resistant (GR) cell lines. We hypothesized that aberrant accumulation of cholesteryl ester (CE) regulated by ACAT-1 could modulate GR in BTC. METHODS: CE accumulations were measured in human BTC cell lines, and the relationships between CE levels, ACAT-1 expressions, and gemcitabine sensitivity were analyzed. We performed a small-interfering RNA (siRNA)-mediated knockdown and biochemical inhibition of ACAT-1 in BTC cell lines and alterations of gemcitabine sensitivity were evaluated. To evaluate the clinical significance of ACAT-1 in regard to GR, immunohistochemistry was performed and ACAT-1 expressions were analyzed in resected BTC specimens. RESULTS: CE levels were correlated with ACAT-1 expressions and GR in four human BTC cell lines. siRNA-mediated knockdown of ACAT-1 in two independent GR cell clones as well as ACAT-1 inhibitor treatment significantly increased gemcitabine sensitivity; knockdown of ACAT-1: 5.63- and 8.02-fold; ACAT-1 inhibitor: 8.75- and 9.13-fold, respectively. ACAT-1 expression in resected BTC specimens revealed that the disease-free survival of the ACAT-1 low-intensity group (median 2.3 years) had a significantly better outcome than that of the ACAT-1 high-intensity group (median 1.1 years) under gemcitabine treatment after surgery (*p < 0.05). CONCLUSIONS: Our findings suggest that CE and ACAT-1 might be a novel therapeutic target for GR in BTC.

A novel substrate mimetic inhibitor of PKB/Akt inhibits prostate cancer tumor growth in mice by blocking the PKB pathway.

We describe a novel, potent peptide substrate mimetic inhibitor of protein kinase B (PKB/Akt). The compound selectively kills prostate cancer cells, in which PKB is highly activated, but not normal cells, or cancer cells in which PKB is not activated. The inhibitor induces apoptosis and inhibits the phosphorylation of PKB substrates in prostate cancer cell lines and significantly increases the efficacy of chemotherapy agents to induce prostate cancer cell death, when given in combination. In vivo, the inhibitor exhibits a strong antitumor effect in two prostate cancer mouse models. Moreover, treated animals develop significantly less lung metastases compared to untreated ones, and the effect is accompanied by a significant decrease in blood PSA [prostate-specific antigen] levels in treated animals. This compound and its potential analogues may be developed into novel, potent, and safe anticancer agents, both as stand-alone treatment and in combination with other chemotherapy agents.

Phase I study of amphotericin B colloidal dispersion for the treatment of invasive fungal infections after marrow transplant.

Amphotericin B colloidal dispersion (ABCD; Amphocil) was evaluated in a phase I dose-escalation study in 75 marrow transplant patients with invasive fungal infections (primarily Aspergillus or Candida species) to determine the toxicity profile, maximum tolerated dose, and clinical response. Escalating doses of 0.5-8.0 mg/kg in 0.5-mg/kg/patient increments were given up to 6 weeks. No infusion-related toxicities were observed in 32% of the patients; 52% had grade 2 and 5% had grade 3 toxicity. No appreciable renal toxicity was observed at any dose level. The estimated maximum tolerated dose was 7.5 mg/kg, defined by rigors and chills and hypotension in 3 of 5 patients at 8.0 mg/kg. The complete or partial response rate across dose levels and infection types was 52%. For specific types of infections, 53% of patients with fungemia had complete responses, and 52% of patients with pneumonia had complete or partial responses. ABCD was safe at doses to 7.5 mg/kg and had tolerable-infusion-related toxicity and demonstrable antifungal activity.

Cholesterol sulfate, a second messenger for the eta isoform of protein kinase C, inhibits promotional phase in mouse skin carcinogenesis.

Cholesterol sulfate is a second messenger for the eta isoform of protein kinase C mediating squamous differentiation. We found that cholesterol sulfate inhibited the promotional phase of skin carcinogenesis in female CD-1 mice, which was initiated by 100 micrograms 7,12-dimethylbenz[a]-anthracene and promoted by a single application of 10 micrograms 12-O-tetradecanoylphorbol-13-acetate, followed by repeated applications of 10 micrograms mezerein once a week for 19 weeks. Cholesterol sulfate, when applied topically at a dose of 400 micrograms (820 mumol) 10 min before treatment with the promoters, markedly suppressed tumor formation, resulting in decrease of 56% in the incidence of tumor-bearing mice, 81% in the number of tumors/mouse, and 60% in the size of tumors at 20 weeks of the promotion. This inhibition was not due to elimination of the initiated cells. Treatment with the parental cholesterol at a dose of 320 micrograms (820 mumol), which does not activate the eta isoform, did not inhibit tumor promotion. Repeated treatment with cholesterol sulfate induced scaling of skin at the site of application. Cholesterol sulfate, unlike most inhibitors of tumor promotion, did not inhibit induction of ornithine decarboxylase and hyperplasia in mouse epidermis caused by topical treatment with 12-O-tetradecanoylphorbol-13-acetate. These findings suggest that cholesterol sulfate inhibits tumor promotion by stimulating a differentiation pathway mediated by the eta isoform of protein kinase C.

Treatment of invasive aspergillosis.

Invasive aspergillosis is generally a life-threatening invasive opportunistic mycosis affecting principally the upper and lower respiratory tract. Therapeutic response rates vary considerably from one host group to another with particularly high mortality rates in bone marrow transplant, liver transplant and patients with aplastic anaemia or AIDS. Only two drugs are useful for therapy, amphotericin and itraconazole. Recent advances in the formulation of amphoterin B (AmBisome and Amphocil) have resulted in intravenous preparations with lower toxicity, particularly nephrotoxicity, but it has yet to be shown that they have an increased therapeutic index for the treatment of invasive aspergillosis. Itraconazole can only be used orally and in some particularly high-risk or critically ill patients adequate serum concentrations cannot be achieved. The addition of flucytosine or rifampicin to amphotericin B therapy has, at best, only a marginal benefit. Surgery is essential for some manifestations of invasive aspergillosis. This article reviews therapeutic strategies including criteria for initiation of therapy, combination and sequential therapy, duration of therapy and secondary prophylaxis and indications for surgery in invasive aspergillosis.

Enhanced antigenicity of autologous leukemia cells enriched with cholesterylhemisuccinate.

Cholesterylhemisuccinate (CHS) has been used by different authors to enhance the antigenicity of tumor cells in human and in animals. In the present study, leukemic cells isolated mainly from patients with chronic myeloid and chronic lymphocytic leukemia were incubated for 90 min at 37 degrees in PBS containing 3.5% polyvinylpyrrolidone, 0.5% glucose, 1% human serum albumin and 150 micrograms/ml CHS 5 x 10(6) pretreated, irradiated, autologous leukemic cells were tested for their ability to elicit a delayed type hypersensitivity skin reaction. The negative controls included CHS-treated and untreated non-leukemic autologous blood cells as well as substance controls. Recall antigens such as tetanus, diphtheria and tuberculine included in the Multitest Mérieux served as positive controls. Results from 28 patients and 44 skin tests are presented. 19 of 23 patients tested under the best of experimental conditions generated a positive skin reaction with the CHS-treated autologous leukemic cells. Less than 12% of the patients reacted with any of the above negative controls. Under the same conditions, more than 90% of the CLL patients were reactive. 5 CLL patients were selected for a treatment including 6 weekly injections of 5 x 10(6) to 5 x 10(8) autologous CLL cells. 3 patients experienced an up to 60% reduction of their initial leukocyte counts. In one patient, the leukocyte counts continued to decrease more than 6 months after the treatment was completed. One patient showed no response and in one patient with increasing leukocytes this increase could not be reversed. The results are discussed in terms of earlier observations that membrane sterol content influences the presentation of cell surface proteins.

Active immunotherapy of human solid tumor with autologous cells treated with cholesteryl hemisuccinate. A Phase I study.

A marked increase in specific immunogenicity of tumor cells is induced upon incorporation of cholesteryl hemisuccinate (CHS) into the cell membrane, which presumably promotes the expression of latent tumor-associated antigens. Immunotherapy with CHS-treated and irradiated tumor cells as vaccine was found to be very effective in various murine experimental tumors and in eliciting delayed-type hypersensitivity in cancer patients. Based on these findings, we have carried out a Phase I study on 21 patients with solid tumor who had exhausted standard therapeutic options. All participating patients were examined by conventional physical and clinical tests prior and during the study. The immunotherapy regimen for most patients consisted of an intramuscular injection of 2 X 10(7) CHS treated and irradiated autologous tumor cells given at 2-week intervals. Variations on this regimen were mostly due to the lack of sufficient number of cells. None of the patients displayed evidence of toxicity or any other local or systemic adverse effects. In seven patients, regression of tumor mass was observed. In six of nine patients who were tested for delayed-type hypersensitivity against their CHS treated tumor cells, a significant increase in skin reaction was observed after immunotherapy. The lack of any adverse reaction in this treatment, in addition to the observed positive clinical and immunologic response in advanced cancer patients, indicate a safe therapeutic potency which is planned to be investigated in the subsequent clinical studies.

Active immunization of hamsters against pancreatic carcinoma with lipid-treated cells or their shed antigens.

Increasing membrane lipid microviscosity of cells by treatment with cholesterol esters such as cholesterol hemisuccinate (CHS) enhances immunogenicity, probably by exposure of cryptic membrane antigens. Transplantable pancreatic carcinoma lines CBP and LSP-1 grown in inbred hamsters were tested for immunogenicity after CHS treatment. Tumor cells were incubated in CHS to rigidify cell membranes, and they were irradiated and injected i.p. into syngeneic hamsters. Incubation media after CHS treatment, considered to contain shed antigens due to hyperrigidification, were also used for immunization. Two identical immunizations using 10(7) cells or incubation media were performed 14 days apart. In control experiments, non-CHS-treated, irradiated cells were injected. Immunizations were performed using both syngeneic and allogeneic cells and supernatants for both the CBP and LSP-1 systems for specificity experiments. The degree of immunization in the treated hamsters was assessed by the response to a subsequent s.c. challenge with viable tumor cells given 7 days following the last immunization. For the CBP pancreatic cancer line, CHS treatment increased tumor immunogenicity significantly, as demonstrated by diminished tumor growth rate and by increased duration of survival after challenge. With the LSP-1 pancreatic tumor, immunization with CHS-treated cells showed no enhancement of immunogenicity. However, immunization with supernatant of CHS-treated cells resulted in a significant delay of tumor growth and increased survival, suggesting immunization by shed antigens in the CHS incubation medium. Use of CHS-treated cells or shed antigeneic material could be of potential value as a method of active immunotherapy.

Regulation of membrane function by lipids; implications for tumor development.

Increase in the lipid fluidity of membranes increases the turnover number of each diffusible unit, but in general decreases its accessibility to ligand binding. As a result, many membranal functions reach a maximal activity at a specific lipid fluidity. These effects of membrane fluidity bear direct implications on tumor development with significant clinical potential. Two main avenues, by which lipid manipulation could be applied in cancer treatment are now being studied in experimental animals and cancer patients. In the first, the immunogenicity of tumor cells in syngeneic and autologous systems increase upon increase of the membrane microviscosity which is in line with the findings that membrane antigens become more exposed upon such treatment. Irradiated tumor cells with increased membrane microviscosity (e.g. by incorporation of cholesteryl hemisuccinate, CHS) thus act as strong and specific vaccine against the viable untreated tumor cells of the same kind. The second lipid manipulation relates to restoration of suppressed immune competence. A special mixture of lipids (Active Lipid, AL) designed to efficiently fluidize cell membranes was found to restore various immunological functions of leukocytes from cancer patients. The combination of augmentation of tumor immunogenicity and restoration of immune functions by such lipid manipulations is expected to constitute an innocuous active immunotherapy regimen for cancer treatment.