Ichiro Tsujino

Effect of Tween-80 on cell killing by etoposide in human lung adenocarcinoma cells.

Purpose: The non-ionic detergent Tween-80, a surface-active agent, has been shown to modulate the cytocidal effect of certain antitumor agents. In the present study, we sought to determine whether or not Tween-80 could enhance the antitumor effect of etoposide (VP16) in human lung cancer cells in vitro. Methods: Survival fractions were measured by growth inhibiton assays of PC14, H69, KB, and PC14/CDDP (the corresponding cisplatin-resistant subline of PC14) cells. An in vitro clonogenic assay of PC14 and PC14/CDDP cells was undertaken after incubation for 10–12 days in RPMI-1640 medium with 20% fetal calf serum and 1.72% methyl cellulose, plus continuous exposure to VP16 with Tween-80. We also investigated the direct toxicity of Tween-80 to PC14 and PC14/CDDP cells using a clonal assay. The intracellular accumulation of VP16 was further analyzed using [3H]VP16 in PC14, PC14/CDDP, A549, KB and H69 cells, and compared with that of daunorubicin (DNR), a hydrophilic anticancer agent, using [3H]DNR in PC14, A549 and KB cells. Results: It was found that PC14/CDDP had collateral sensitivity to VP16 and Tween-80 markedly enhanced the killing effect of VP16 not only of PC14 cells but also of PC14/CDDP cells while exerting little cytotoxic effect. Moreover, Tween-80 increased the intracellular accumulation of VP16 in PC14, PC14/CDDP and A549 cells, and not in KB and H69 cells. Tween-80 did not increase the intracellular DNR levels in PC14, A549 and KB cells. Conclusions: Tween-80 was shown to potentiate the cytotoxicity of VP16 against several human lung adenocarcinoma cells by increasing the accumulation of VP16 in vitro. Tween-80-mediated sensitization of lung adenocarcinoma cells to VP16 is considered to be related to both the characteristics of the cell membrane in adenocarcinoma cells and the lipotropic properties of VP16. These results suggest that this combination might have the potential to improve the therapeutic index of VP16 in human lung adenocarcinoma.

Non-ionic detergent Tween 80 modulates VP-16 resistance in classical multidrug resistant K562 cells via enhancement of VP-16 influx.

The non-ionic detergent Tween 80, which is used as a solvent for lipophilic drugs such as VP-16 and Taxotere, was found to reverse VP-16 resistance of the P-glycoprotein-associated multidrug resistance phenotype via increasing VP-16 influx. In adriamycin-resistant human chronic myelogenous leukemia K562 cells (K562/ADM), which overexpress mdr1 mRNA, the accumulation of VP-16 was only about 10% that in wild-type K562 cells. Tween 80 enhanced VP-16 accumulation in K562/ADM cells but did not influence VP-16 accumulation in parental K562 cells. VP-16 efflux was rapid and similar in both sensitive and resistant cell lines and was not blocked by Tween 80 or verapamil. Under glucose-free conditions, VP-16 accumulation in K562/ADM cells was only half of that in K562 cells. Tween 80 increased VP-16 accumulation in K562/ADM cells in glucose-free medium. In growth inhibition assay, Tween 80 reversed K562/ADM sensitivity to VP-16 without cell damage. Taken together, Tween 80 reverses VP-16 sensitivity in multidrug-resistant K562 cells by increasing influx, which is considered to be the primary mechanism of VP-16 resistance in K562/ADM cells.

Postirradiation Hyperthermia Selectively Potentiates the Merocyanine 540-Sensitized Photoinactivation of Small Cell Lung Cancer Cells¶

Lung cancer has long been considered a disease that might benefit from the dose escalation of radio/chemotherapy afforded by a stem cell transplant. However, the clinical experience with high‐dose chemotherapy and autologous bone marrow transplantation in lung cancer has been disappointing, with most trials showing little or no improvement in long‐term survival. Unfortunately, lung cancer has a tendency to metastasize to the bone marrow, and lung cancer cells are known to circulate in the peripheral blood. Therefore, there is concern that autologous stem cell grafts from lung cancer patients may reinoculate recipients with live tumor cells. Photochemical purging of stem cell grafts with Merocyanine 540 (MC540) is highly effective against a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Most solid tumor cells (including lung cancer cells), however, are only moderately sensitive or refractory to MC540‐mediated photodynamic therapy (PDT). We report here that postirradiation hyperthermia (≤42°C, 3 h) potentiates the MC540‐mediated photoinactivation of both wild‐type (H69) and cisplatin‐resistant mutant (H69/CDDP) small cell lung cancer cells by several orders of magnitude, while only minimally enhancing the depletion of normal human granulocyte/macrophage progenitor cells. Our data suggest that postirradiation hyperthermia provides a simple and effective means of extending the utility of MC540‐PDT to the purging of stem cell grafts contaminated with lung cancer and possibly other solid tumor cells.

Elemental Selenium Generated by the Photobleaching of Seleno-Merocyanine Photosensitizers Forms Conjugates with Serum Macro-Molecules That are Toxic to Tumor Cells

Abstract Elemental selenium generated by the photobleaching of selenomerocyanine dyes forms conjugates with serum albumin and serum lipoproteins that are toxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic stem and progenitor cells. Serum albumin and lipoproteins act as Trojan horses that deliver the cytotoxic entity (elemental selenium) to tumor cells as part of a physiological process. They exploit the fact that many tumors have an increased demand for albumin and/or low-density lipoprotein. Se(0)-protein conjugates are more toxic than selenium dioxide, sodium selenite, selenomethionine, or selenocystine. They are only minimally affected by a drug resistance mechanism, and they potentiate the cytotoxic effect of ionizing radiation and several standard chemotherapeutic agents. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood. Currently available data are consistent with the notion that Se(0)-protein conjugates act as air oxidation catalysts that cause a rapid depletion of intracellular glutathione and induce apoptosis. Drugs modeled after our Se(0)-protein conjugates may prove useful for the local and/or systemic therapy of cancer.

Potentiation of the Antitumor Effect of Merocyanine 540–mediated Photodynamic Therapy by Amifostine and Amphotericin B

Abstract Leukemia and lymphoma cells are much more sensitive to Merocyanine 540 (MC540)–mediated photodynamic therapy (PDT) than normal pluripotent hematopoietic stem cells and normal colony forming unit–granulocyte/macrophage progenitors (CFU-GM). By contrast, most solid tumor cells are only moderately sensitive to MC540-PDT. The limited activity against solid tumor cells has detracted from MC540s appeal as a broad-spectrum purging agent. We report here that noncytotoxic concentrations of amifostine (Ethyol, Ethiofos, WR-2721) and amphotericin B used either alone or in combination potentiate the MC540-sensitized photoinactivation of leukemia cells, wild-type small cell lung cancer cells and cisplatin-resistant small cell lung cancer cells. Amphotericin B also enhances the MC540-sensitized photoinactivation of normal CFU-GM, whereas amifostine protects CFU-GM against the cytotoxic action of MC540-PDT. The yield of CD34-positive normal hematopoietic stem and progenitor cells is only minimally diminished by pretreatment with amifostine, amphotericin B or combinations of amifostine plus amphotericin B. Purging protocols that combine MC540-PDT with amifostine or with amifostine plus amphotericin B could offer a simple and effective approach to the purging of autologous stem cell grafts that are contaminated with solid tumor cells or the purging of stem cell grafts from heavily pretreated leukemia patients that contain reduced numbers of normal stem and progenitor cells and, therefore, can ill afford additional losses caused by purging.

Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC.

Examination of expandable metallic stent removed at autopsy

Objective:  We evaluated the damage to expandable metallic stents (EMS) based upon analysis of EMS removed at autopsy.

Photochemically generated elemental selenium forms conjugates with serum proteins that are preferentially cytotoxic to leukemia and selected solid tumor cells.

The objective of this study was to determine if and how photoproducts contribute to the antitumor effect of merocyanine‐mediated PDT. A panel of barbituric, thiobarbituric and selenobarbituric acid analogues of Merocyanine 540 was photobleached, and the resulting photoproducts were characterized by absorption, fluorescence emission, mass, energy dispersive X‐ray, and X‐ray photoelectron spectroscopy and tested for cytotoxic activity against tumor cell lines and freshly explanted bone marrow cells. While all dyes were readily photobleached, only photoproducts of selone dyes showed cytotoxic activity. One‐hour incubations with micromolar concentrations of selone‐derived photoproducts were sufficient to reduce leukemia/lymphoma cells ≥10 000 fold, whereas preserving virtually all normal CD34‐positive bone marrow cells. Of six multidrug‐resistant tumor cell lines tested, five were as sensitive or more sensitive to photoproducts than the corresponding wild‐type lines. Physicochemical characterizations of the cytotoxic activity indicated that it consisted of conjugates of subnano particles of elemental selenium and (lipo)proteins. The discovery of cytotoxic Se‐protein conjugates provides a rare example of photoproducts contributing substantially to the antitumor effect of PDT and challenges the long‐held view that Se in oxidation state zero is biologically inert. Agents modeled after our Se‐protein conjugates may prove useful for the treatment of leukemia.

Genetic Variability in the Response of Normal Murine Hematopoietic Progenitor Cells to Extracorporeal Photochemotherapy

Abstract Normal hematopoietic progenitor cells from 129S6/SvEv mice are substantially less sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than hematopoietic progenitors from sex- and age-matched C57BL/6 mice. When exposed to a combination of MC540 and light commonly used for the extracorporeal purging of hematopoietic stem cells, granulocyte/macrophage progenitors (CFU-GM) from C57BL/6 mice are depleted 7.9-fold whereas CFU-GM from 129S6/SvEv and (C57BL/6 × 129S6/SvEv) F1 mice are depleted 1.4- and 2-fold, respectively. The same rank order of sensitivity is also found with regard to unipotent progenitors of granulocytes and macrophages and with regard to early and late erythroid progenitors. The resistance of hematopoietic progenitors from 129S6/SvEv mice to MC540-PDT appears to be the result of reduced dye binding rather than the result of high levels of intracellular glutathione. These findings have practical implications for the design of preclinical tests of PDT in animal models. They may also provide a useful tool for future investigations into the molecular determinants of sensitivity to MC540-PDT.

Nicotine-Mediated Ca(2+)-Influx Induces IL-8 Secretion in Oral Squamous Cell Carcinoma Cell.

Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin‐8 (IL‐8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine‐mediated IL‐8 induction in OSCC was investigated. Augmented IL‐8 secretion by Ca9‐22 cells was blocked by the NF‐κB inhibitor L‐1‐4′‐tosylamino‐phenylethyl‐chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)‐specific inhibitor α‐bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre‐incubating the cells with inhibitors against mitogen‐activated protein kinase (MEK), protein kinase C (PKC), and Ca2+/calmodulin‐dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine‐mediated IL‐8 secretion. Pre‐treatment of the Ca9‐22 cells with the Ca2+ chelator BAPTA‐AM drastically inhibited IL‐8 secretion. Although nicotine stimulation induced the phosphorylation of the NF‐κB p65 subunit, pre‐treatment with BAPTA‐AM was found to inhibit this activity significantly. CaMK II‐dependent p65 phosphorylation was confirmed by pre‐incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca2+ influx, which results in the activation and phosphorylation of CaMK II and NF‐κB p65, respectively. Nicotine‐mediated IL‐8 induction should be a trigger for the initiation of various diseases. J. Cell. Biochem. 117: 1009–1015, 2016.