Effect of tumor necrosis factor and interferon gamma on human renal carcinoma cell line growth

Four human renal carcinoma cell lines (786-0, CaKi-1, TK-10 and TK-164) were studied in vitro for susceptibility to human recombinant interferon-gamma (IFN gamma) and/or human recombinant tumor necrosis factor (TNF). All four cell lines tested demonstrated a dose dependent sensitivity to the cytotoxic effects of TNF alone. The degree of sensitivity varied for each cell line. IFN gamma alone also mediated a dose dependent antiproliferative effect in three of the four cell lines. Combinations of IFN gamma and TNF produced diverse inhibitory effects. Preincubation of the cells for various time intervals with noninhibitory concentrations of IFN gamma prior to the addition of TNF resulted in distinct effects for each cell line. Pretreatment enhanced the cytotoxic effect of TNF for some cell lines; however, others became less susceptible and some demonstrated enhanced proliferation in the presence of both agents. Optimal pretreament times also varied from cell line to cell line. These results demonstrate variability in responsiveness by renal carcinoma cell lines to combination IFN gamma and TNF treatments and suggest that timing as well as concentration may be important in in vivo therapy.     

Type I interferons in the treatment of pancreatic cancer: mechanisms of action and role of related receptors

OBJECTIVE: We evaluated the role of type I interferons (IFNs) and IFN receptors in the regulation of cell growth in 3 human pancreatic adenocarcinoma cell lines (BxPC-3, MiaPaCa-2, and Panc-1). BACKGROUND: Chemotherapy and radiotherapy have a marginal role in the management of pancreatic adenocarcinoma. The addition of IFN-alpha showed promising results in early clinical trials. METHODS: Cell proliferation and apoptosis were evaluated by DNA measurement and DNA fragmentation, respectively. Type I IFN receptor (IFNAR-1 and IFNAR-2 subunits) was determined by quantitative RT-PCR and immunocytochemistry. Cell cycle distribution was evaluated by propidium iodide staining and flow-cytometric analysis. RESULTS: The incubation with IFN-beta for 6 days showed a potent inhibitory effect on the proliferation of BxPC-3 (IC(50), 14 IU/mL) and MiaPaCa-2 (IC(50), 64 IU/mL). The inhibitory effect of IFN-beta was stronger than IFN-alpha in all 3 cell lines and mainly modulated by the stimulation of apoptosis, although cell cycle arrest was induced as well. The expression of the type I IFN receptors was significantly higher in BxPC-3 (the most sensitive cell line to IFN) and mainly localized on the membrane, whereas in Panc-1 (the most resistant cell line) about 60% to 70% of cells were negative for IFNAR-2c with a mainly cytoplasmic staining for IFNAR-2c. CONCLUSION: The antitumor activity of IFN-beta is more potent than IFN-alpha in pancreatic cancer cell lines through the induction of apoptosis. Further studies should investigate in vivo whether the intensity and distribution of IFNAR-1 and IFNAR-2c may predict the response to therapy with IFN-alpha and IFN-beta in pancreatic cancer.     

Influence of interferon alfa-2c on the kinetics of spontaneous cell-mediated cytotoxicity in Urothelial carcinoma in vivo and in vitro

Summary It was demonstrated that patients with superficial bladder cancer displayed a raised spontaneous cellmediated cytotoxicity (SCMC) compared to patients with advanced bladder cancer and healthy control subjects. By use of recombinant interferon alfa-2c, the activity of the spontaneous cell-mediated cytotoxicity at the level of the individual cell could be increased both in vitro and in vivo. In vitro, this was the case in patients with superficial bladder cancer as well as in patients with advanced bladder carcinoma, and in healthy control subjects. The kinetics of cytolysis were not affected by recombinant human interferon (rHu IFN) alfa-2c. After in-vivo application of rHu IFN, there was an elevation of the target binding cells (TBC) and the number of active natural killer (NK) cells within 24 h, but this was only detected for a brief period of time.     

Differential antiproliferative activities of alpha- and gamma-interferon and tumor necrosis factor alone or in combinations against two prostate cancer xenografts transplanted in nude mice

We have investigated the antiproliferative effects of recombinant human alpha- and gamma-Interferon (IFN) and recombinant human Tumor Necrosis Factor alpha (TNF) against the hormone-independently growing PC3 and DU145 prostatic tumor lines. Subcutaneous, peritumoral administration of the drugs was started 24 hours after subcutaneous implantation of 1–2 mm³ tumor pieces. IFN was given three times per week and TNF five times per week. IFN-alpha (dose-range 0.5–5 ng/gram bodyweight) had significant growth-inhibiting effects against the PC3 tumor, but showed no significant antitumor effects against the DU145 tumor. IFN-gamma monotherapy (dose-range 8–80 ng/gram bodyweight) was less effective than IFN-alpha. 500 ng/gram TNF produced growth inhibition of both tumors, whereas the lower dose (50 ng/g) was only effective against the PC3 tumor. IFN-alpha and -gamma combination treatment had significant antiproliferative effects against the PC3 tumor, but not against the DU145 tumor. Combinations of IFN-alpha and TNF were very effective against both xenografts; some combinations resulted in complete growth inhibition. IFN-gamma and TNF combinations also showed significant antitumor effects against both tumor lines. We therefore conclude that cytokine combination treatment may provide a new approach in the treatment of hormone-escaped prostatic tumors.     

Effects of bacillus Calmette-Guèrin and interferon alpha-2B on cytokine production in human bladder cancer cell lines

PURPOSE: To determine the effects of live BCG, autoclaved BCG and interferon alpha-2b on cytokine production in human bladder cancer cell lines. MATERIALS AND METHODS: The release of nine cytokines from the human bladder cancer cell lines, RT4, RT112, SD, MGH and J82, was measured by ELISA assay. The mRNA level of IL-6 and GM-CSF was determined by RT-PCR. RESULTS: BCG and/or interferon alpha-2b differentially increased IL-1beta, IL-6, IL-8, GM-CSF and TNF-alpha production in the bladder cancer cells. High grade cell lines were more responsive to BCG whereas low grade lines were more sensitive to interferon alpha-2b. This correlated with cytotoxicity and growth inhibition induced by these agents. BCG could also induce low levels of IFN-alpha production in all the cell lines. Compared with live BCG, autoclaved BCG had no antiproliferative effect on MGH cells and was less effective in stimulating the production of IL-6, IL-8 and GM-CSF. However, autoclaved BCG was as effective as live BCG in inhibiting growth and stimulating IL-6 and TNF-alpha production of J82 cells. The combination of BCG and interferon alpha-2b also completely suppressed TGF-beta1 production in the MGH and RT112 cell lines. CONCLUSIONS: The combination of BCG and interferon alpha-2b has additive effects in cytokine production from bladder cancer cells. This correlates with cytotoxicity and growth inhibition induced by these agents.     

Interferon receptors and the caspase cascade regulate the antitumor effects of interferons on human pancreatic cancer cell lines

BACKGROUND: Interferons (IFNs) have antiproliferative effects on tumor cells. The apoptotic effects and sensitization to chemotherapy conferred by IFN therapy, however, are not clearly understood. The aims of the present study were to explore the apoptotic effects of IFNs in human pancreatic cancer cell lines and to attempt to define their ability to synergistically enhance sensitivity to 5-fluorouracil (5-FU) and gemcitabine, a mechanism that depends on the expression of IFN receptors. METHODS: Human pancreatic cancer cells were cultured alone or in combination with the chemotherapeutic agents 5-FU and gemcitabine. Differential dosages of IFN-alpha, -beta, and -gamma were also added to the cell lines concomitantly during a period of 24 to 96 hours. The cell line viability and effects of treatment were examined using the methylthiazol tetrazolium assay and single-stranded DNA apoptosis assay. The expression of IFN receptors was determined using immunohistochemistry. Caspase-8 inhibitor was used to block the caspase cascade. RESULTS: The antiproliferative and apoptotic effects of IFNs were most profoundly demonstrated on those cells that expressed the respective IFN receptor. The apoptotic effects provided by the interferons, however, were blocked by caspase-8 inhibition. The addition of IFNs significantly enhanced the cytotoxic effects of 5-FU and gemcitabine in those cell lines that expressed the corresponding IFN-alpha, -beta, or -gamma receptors. CONCLUSIONS: This study on pancreatic cancer cell lines has demonstrated that IFNs mediate apoptosis through IFN receptors and the caspase cascade. Enhanced cytotoxicity occurred when IFNs were combined with 5-FU and gemcitabine.     

The effects of immuno-chemotherapy, hyperthermia and irradiation on the growth of human renal cell carcinoma

In order to investigate more effective treatments for advanced renal cell carcinoma, experiments were performed as to inhibitory effects on the growth of KPK 1 cells derived from a human renal carcinoma. IFN-beta, IFN-gamma, vinca alkaloids (VBL, VCR, VDS), hyperthermia and irradiation inhibited the growth of KPK 1 cells. Both IFN-beta and IFN-gamma showed concentration dependent and time dependent inhibitory effects, and IFN-beta was significantly more intensive in action than IFN-gamma. All of vinca alkaloids inhibited the cell growth remarkably but there was no difference among these three drugs. Hyperthermia was inhibitory over 43 degrees C and irradiation showed a remarkable inhibition at not less than 500 rads. But IFN-alpha and hormones had no inhibitory effect. These results imply that multidisciplinary treatments in combination of immuno-chemotherapy with IFN-beta, IFN-gamma or vinca alkaloids, hyperthermia and irradiation should be applied for advanced renal cell carcinoma.     

Immunomodulatory effects of interferons on target human gliosarcoma cells in the tumor-specific CTL- and LAK-mediated cytolysis]

We compared the regulatory effects of interferon (IFN)-beta and IFN-gamma on the susceptibility of a human gliosarcoma line GI-1 to the attack of autologous cloned tumor-specific cytotoxic T-lymphocytes (CTL) and lymphokine-activated killer (LAK) cells. Preincubation of GI-1 cells with IFN-gamma caused augmented susceptibility to the cytotoxic attack of two autologous CTL clones, whereas IFN-beta exhibited no such marked effect. On the other hand, preincubation with either IFN-beta or IFN-gamma made the GI-1 cells resistant to the attack of autologous LAK cells. Both IFNs augmented the surface expression of HLA class-I molecules on GI-1 cells. A monoclonal anti-HLA class-I antibody blocked the cytolysis by one CTL clone, but not by the other one. These results suggest that IFN-gamma exerts some different effect (s) from that of IFN-beta on the target GI-1 cells in their susceptibility to the CTL-mediated cytolysis, and that recognition mechanisms of target cells by the CTL are different from those by LAK cells. This draws our attention to IFN administration in adoptive immunotherapy against brain tumors using CTLs and LAK cells.     

Experimental studies on the combined effects of alpha and gamma interferons against human tumor xenografts transplanted into nude mice

The antitumor activities, resulting from the combined treatment of leukocyte interferon (IFN-alpha), with recombinant human immune interferon (IFN-gamma), against human tumor xenografts in nude mice, were studied. Nine human tumor xenografts, (7 from gastric carcinoma, 1 from gallbladder carcinoma and 1 from breast carcinoma), were serially transplanted into nude mice for the purpose of this experiment. Each human tumor xenograft was inoculated subcutaneously into BALB/c nu/nu nude mice and treatment was started after the estimated tumor had reached 100-300 mg. IFN was administered intramuscularly at a schedule of qd X 14. Treatment with either IFN-alpha or IFN-gamma alone, did not produce any antitumor effect against the various human tumor xenografts, however the combination of IFN-alpha with IFN-gamma resulted in achieving significant antitumor effects against the various human tumors. Inhibition of tumor growth was observed in 7 of the 9 tumors (77.8 per cent), and regression of the tumor was noted in 5 of the 9 tumors (55.6 per cent).     

Hyperthermia with simultaneous administration of interferon using established human renal carcinoma heterotransplanted in nude mice

Using human renal carcinoma heterotransplanted in nude mice, the effects of the combined use of hyperthermia and interferon (natural human interferon-alpha; IFN-alpha, and recombinant interferon-gamma; IFN-gamma) were examined. The hyperthermic device used was the Clini-Therm, Mark VII, 915 MHz, microwave. The administration of interferon was started immediately before hyperthermia and carried out on consecutive days. The combined use of hyperthermia and IFN was compared with hyperthermia and with IFN alone. The combined use of hyperthermia and IFN-alpha was shown to have significant anti-tumour effects compared with the former or latter alone. Of 10 nude mice (10 implanted tumours) used for the experiment, 5 showed complete disappearance of the implanted tumours and the others showed prolongation of survival. Combined treatment with IFN-gamma and hyperthermia showed no significant effect when compared with other treatment groups. Combined treatment with local hyperthermia and IFN-alpha may have some application to the clinical management of renal carcinoma.     

The development of liposomes containing interferon alpha for the intravesical therapy of human superficial bladder cancer

Current therapy of human superficial bladder cancer includes the intravesical administration of antitumor drugs and immunomodulators. The purpose of these studies was to determine whether phospholipid liposomes that bind to human bladder cancer cells can improve the delivery of interferon alpha (IFN-alpha) to neoplastic urothelium. The antiproliferative activity of free IFN-alpha and IFN-alpha encapsulated in liposomes was assessed in vitro against the human transitional cell carcinoma line 253J. The cells were exposed to free and liposome-encapsulated IFN-alpha for short periods ranging from 30 minutes to four hours, and inhibition of cell growth was determined three days later. The production of greater than 25 percent cytostasis of 253J cells by free IFN-alpha required four hours of continuous exposure. In contrast, IFN-alpha encapsulated in liposomes produced 35 percent and 60 percent cytostasis after a 30-minute and four-hour exposure, respectively. Liposome-encapsulated IFN-alpha was also effective (50 percent cytostasis) against a subline of 253J cells selected for resistance against free IFN-alpha. Liposomes containing IFN-alpha were stable in the presence of human urine. In vivo studies in mice showed that intravesical administration of radiolabeled IFN-alpha or radiolabeled liposomes did not yield significant systemic absorption and deposition in distant organs. Collectively, these results suggest that the encapsulation of IFN-alpha within multilamellar liposomes may augment its antiproliferative activity, overcome some forms of tumor cell resistance to IFN-alpha, and prove useful for intravesical therapy of superficial bladder cancer.     

Antiproliferative activities of interferons against human bladder carcinoma cell lines in vitro

The antiproliferative effect of interferons against 5 human bladder carcinoma cell lines, RT112, T24, RT4, 647V and HT1197, was determined in vitro. Each of these human bladder carcinoma cell lines except 647V was sensitive to human interferons in liquid media. The antiproliferative effect of interferons was observed only upon continuous exposure, not after 1 hour. Partially purified, naturally produced interferon beta was more inhibitory of cell growth than naturally produced interferon alpha. Interferon alpha 54, 76, 61, 6L and 1 purified to homogeneity were as effective as naturally produced, partially pure interferon alpha. Although interferon beta, produced by recombinant DNA technology and purified to homogeneity, was not equivalent in effectiveness to naturally produced interferon beta, its antiproliferative activity was greater than interferon alpha 54 for 3 of 4 cell lines tested. Antimitotic effects may underlie, at least in part, the potential therapeutic activity of interferons for bladder carcinoma.     

Biological potentials of interferons relevance in the systemic treatment of superficial bladder carcinoma

Summary Interferons (IFN) are a class of glycoproteins which have antiviral, antiproliferative and immunomodulating properties. Three major classes of IFN are characterized today on the basis of antigenic and physical-chemical properties. They modulate various immunological functions and can be characterized as biological response modifiers. In addition, they have direct cytotoxic effects on tumor cells. Since 1978, interferons have been in clinical use as antineoplastic agents in patients with superficial bladder tumors. The first reports on the treatment of superficial bladder tumors with natural IFN preparations offered encouraging results. Recently published data with systemically applied recombinant IFN alpha-2, however, did not confirm the good results previously reported. One of the reasons for these conflicting results might be the different types of IFN alpha used. Since laboratory results have demonstrated that reduction of the tumor cell multiplication rate can be influenced by the concentration of IFN, topical application, e.g. in the therapy of superficial bladder carcinoma, may offer more promising results in comparison to systemic application. Further knowledge on the immunomodulatory and anticellular mechanisms is needed in order to allow a successful application of IFN in the treatment of cancer.     

Antitumor activity of the antimicrobial peptide magainin II against bladder cancer cell lines

OBJECTIVE: Magainin II belongs to a family of antimicrobial peptides and has been shown to exhibit antibiotic activity in a wide range of organisms. Recent studies have also reported a significant antitumor effect of magainin II against various cancer cell lines and tumor mice models. In this study, we evaluated the cytotoxic and antiproliferative potency of magainin II in bladder tumor cells and normal fibroblasts. METHODS: The antiproliferative and cytotoxic effect of magainin II was quantified by colorimetric WST-1-, bromodeoxyuridine (BrdU)-, and lactic dehydrogenase (LDH) assays in three bladder cancer cell lines (RT4, 647V, and 486P) and in the murine fibroblast cell line 3T3 as well as in a primary culture from human fibroblasts. The median inhibitory concentration (IC50) values were determined for each assay, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was used to visualize the morphologic effects of magainin II on bladder tumor cells and fibroblasts. RESULTS: Magainin II inhibited cell proliferation of bladder cancer cells in a dose-dependent manner. The average IC50 of magainin II against all bladder cancer cell lines was 198.1 microM (range, 52.4-484.03 microM) for the WST-1 assay and 75.2 microM (range, 31.0-135.3 microM) for the BrdU assay. The normal murine and human fibroblast cell lines were not affected by magainin II and their IC50 could not be determined at the concentrations of magainin II tested. LDH release was increased in all bladder tumor cell lines in the presence of magainin II, whereas normal fibroblasts showed no cell lysis. SEM demonstrated lethal membrane perforation by peptide pore formation in bladder cancer cells, but not in fibroblasts. CONCLUSION: Magainin II peptide exerts cytotoxic and antiproliferative efficacy by pore formation in bladder cancer cells but has no effect on normal murine or human fibroblasts. Magainin II may offer a novel therapeutic strategy in the treatment of bladder cancer with potentially low cytotoxic effects on normal cells.     

Antiproliferative effect of interferon-alpha on human renal cell carcinoma in clonogenic assay--single and combination effect with cancer chemotherapeutic agent

With human tumor clonogenic assay, the direct antiproliferative activity of recombinant human leukocyte interferon alpha (IFN-alpha) was investigated on human renal cell carcinomas (RCCs), which consisted of a human RCC cell line (ACHN), two human RCC xenografts and fifteen primary RCCs. The combination effect of IFN-alpha with a cancer chemotherapeutic agent was studied, as well, with the assay system. IFN-alpha showed a dose-dependent antiproliferative activity against the human RCCs. The clonal growth of ACHN cell line was inhibited by less than 50% at the concentration of 1,000 IU/ml. Two xenografts had a different sensitivity to IFN-alpha, in which the percent colony formation was less than 20% in RCC-3 at the concentration of 100-100,000 IU/ml, while in RCC-4 more than 50% even at the high concentration of 10,000 IU/ml. In 15 primary tumors obtained at surgery, two types of response to IFN-alpha were demonstrated. One was the response in which the colony formation was inhibited in a dose-dependent manner as an increment of IFN-alpha concentration, and the other in which the colony formation was not sufficiently inhibited even at the high concentration of IFN-alpha. The dose-dependent inhibition of colony formation was demonstrated in 10 out of 15 specimens (66.7%). When the colony formation suppressed to less than 50% of control was considered to be sensitive to IFN-alpha, 6.7% of these 15 primary tumors were sensitive to IFN-alpha at 100 IU/ml, 20.0% at 1,000 IU/ml and 20.0% at 10,000 IU/ml. Combination effects of IFN-alpha and with each of four different cancer chemotherapeutic agents (vinblastine, adriamycin, methotrexate, 5-fluorouracil) were investigated on the ACHN cell line. Every combination type produced a subadditive or synergistic combination effect. In particular, the combination of IFN-alpha with vinblastine of more than 0.1 microgram/ml concentration yielded a combination effect of statistical significance (p less than 0.001). Even against premary tumors, the combination of IFN-alpha with vinblastine showed a synergistic effect in one out of every three tumors. These results suggested that the combination of IFN-alpha with a cancer chemotherapeutic agent would enhance the clinical effect of IFN-alpha alone in only a certain situation.     

Antiproliferative effect of hu-interferon-gamma in 674V and J82 bladder carcinoma cell lines

Summary Hu-IFN-gamma was evaluated in regard to the antiproliferative effect on J82 and 647V bladder cancer cell lines. In addition, the IFN-receptors were determined. There was a significant growth inhibition of J82 as well as 647V at low dose Hu-IFN-g (1 U/ml). The growth inhibition was significantly higher in 647V than in J82. The binding assay for 125J-Hu-IFN-g revealed 870 and 3,000 binding sites for 647V and J82, respectively, indicating that the antiproliferative effect of Hu-IFN-g may not depend on the absolute amount of IFN-receptors, in the two cell lines tested.     

Interferon receptors on the surface of interferon-sensitive and interferon-resistant urothelial carcinomas

Summary In previous investigations [3], it was demonstrated that interferons (IFN) have antiproliferative effects in human urothelial carcinomas. However, appreciable differences were found in the sensitivity of the individual tumors investigated. We therefore examined whether this might be due to a different receptor status of the cells. The IFN-sensitive cell lines RT4 and SD as well as the IFN-resistant cell line 639V were investigated with regard to their IFN receptor status. It was demonstrated that IFN receptors were present on the cell surface in all three urothelial carcinomas investigated. The number of IFN receptors calculated for the IFN-resistant cell line 639V was 4.661 per cell, whereas the IFN-sensitive cell line SD had 4.391 receptors and RT4 had 3.307 receptors. The IFN affinity of the three cell lines tested differed only slightly. Therefore IFN affinity is unlikely to account for their marked differences in IFN sensitivity.This study was performed with financial support from the Wilhelm-Sander Foundation     

Influence of cyclic interferon gamma on lymphocytes and their subpopulations in patients with renal carcinoma

Natural killer (NK) cell stimulation can be repeatedly elicited by cyclic administration of recombinant human interferon (rHu IFN) gamma in patients with metastatic renal cell cancer. In all cycles of treatment, the elevation of the cytolytic activity of the NK cells was statistically significant. The shift of the T4/T8 ratio (T-helper/T-suppressor lymphocytes) a short time after the commencement of IFN application might be a manifestation of the immunostimulation induced.     

Changes in epidermal growth factor receptor expression in human bladder cancer cell lines following interferon-alpha treatment

PURPOSE: We examined the regulation of epidermal growth factor (EGF) receptor (EGFR) expression in human bladder cancer cell lines by interferon-alpha (IFN-alpha), the ability of IFN-alpha to inhibit cell proliferation and the sensitivity of IFN-alpha pretreated cells to EGF. MATERIALS AND METHODS: Cell proliferation was determined using crystal violet colorimetric and clonogenic assays. EGFR expression was measured by flow cytometry using specific antibody or ligand binding approaches. RESULTS: After IFN-alpha (100 IU/ml) treatment cell surface EGFR expression was upregulated in 6 of 11 and down-regulated in 2 of 11 bladder cancer cell lines. The over expression of cell surface EGFR peaked within 48 to 96 hours and increased by 35% to 241% in individual cell lines. High level cell surface EGFR correlated with intracellular EGFR expression. Cell growth inhibition by IFN-alpha coexisted with EGFR over expression in the 6 lines. IFN-alpha treated cells remained sensitive to EGF treatment. CONCLUSIONS: IFN-alpha transiently up-regulates EGFR expression and inhibits in vitro growth in some human bladder cancer cells. IFN-alpha does not prevent EGFR from binding EGF or signal transduction via the EGF-EGFR pathway. This may have clinical implications for improving treatment based on EGFR targeting in select patients with bladder cancer.