Cytotoxic effects of alpha- and gamma-interferon and tumor necrosis factor in human bladder tumor cell lines

We investigated the activity of alpha-interferon (-IFN), gamma-interferon (-IFN) and tumor necrosis factor-alpha (TNF-) in a panel of ten human bladder tumor cell lines. All cytokines were tested at concentrations of 100–10000 U/ml in a clonogenic assay system. We found that -IFN was active against five of the ten lines while -IFN was only active against one line. TNF was active against five of the ten lines. Maximum synergisms were obtained between the -IFN and TNF, occurring in nine of the ten cell lines. We conclude that -IFN and TNF are active as single agents and synergistic when used together in vitro in human bladder tumor cell lines.     

Enhanced anti-tumor effects of recombinant human tumor necrosis factor plus VP-16 on metastatic renal cell carcinoma in a xenograft model

A nude mouse renal subcapsular and subcutaneous implantation xenograft model utilizing the SN12C human renal carcinoma cell line was investigated. In the absence of treatment, renal subcapsular implantation of SN12C resulted in metastatic spread (lung, liver and lymph nodes) and death of all animals. Radical nephrectomy of the tumor-bearing kidney after various periods of tumor implantation demonstrated that surgery alone after 18 days of tumor growth resulted in no statistically significant increase in survival with 100% of the nephrectomized animals succumbing to local recurrence and distant metastases. Recombinant human tumor necrosis factor (rTNF) and VP16 (etoposide), both well known cytotoxic and cytostatic anticancer agents, were tested singly and in combination against this metastatic model of human renal adenocarcinoma. Single agent rTNF or VP16 therapy after radical nephrectomy demonstrated only minimal efficacy with no significant decrease in local recurrence and distant metastases as compared to nephrectomy only control animals. In contrast, the combination of rTNF plus VP16 when given after nephrectomy resulted in a significant decrease in local recurrence and no gross evidence of metastasis in any animal. Subcutaneously growing SN12C tumor nodules were also treated with the same rTNF, VP16 and combination regimens. Regression in tumor size was noted only in the combination treatment group. rTNF or VP16, as single agents, demonstrated only slight growth inhibition that was not statistically significant. These results suggest that by combining TNF plus VP16, a synergistic enhancement of antineoplastic activity against local as well as metastatic human renal cell carcinoma can be produced.     

Enhanced in vivo cytotoxicity of recombinant human tumor necrosis factor with etoposide in human renal cell carcinoma

Summary The combination of tumor necrosis factor (TNF) and etoposide (ETP) was evaluated for potential cytotoxic efficacy against a human renal cell carcinoma xenograft using an in vivo assay employing an athymic mouse host with tumor implanted a the subrenal capsule site. Both antitumor efficacy (relative survival or RTS) and toxicity (weight loss) of TNF and ETP alone and in combination were evaluated. While TNF and ETP alone were mildly inhibitory (RTS 90% and 71%, respectively), the combination caused marked tumor inhibition (45% of controls). Host toxicity encountered with the combination did not exceed the toxicity associated with ETP alone, suggesting that the therapeutic index may have been augmented. It is concluded that enhanced antitumor activity without substantial augmentation of toxicity is observed with this combination, providing a rationale for further evaluation of tumor necrosis factor-based regimens for the treatment of advanced renal carcinoma.Supported by a Merit Review grant, VA Medical Research Service, Durham, NC 27710, USA     

Combined effect of tumor necrosis factor alpha and anticancer chemotherapeutic agents against human renal carcinoma cell lines.

Antitumor effect of recombinant human tumor necrosis factor alpha (TNF) alone or in combination with various anticancer chemotherapeutic agents was tested using an in vitro antiproliferative assay on four human renal carcinoma cell lines (VMRC-RCW, VMRC-RCZ, Caki-1 and A-498). When the dose-dependent effect of TNF was studied, none of the four cell lines showed a 50% or more inhibition even at a concentration of 10,000 U/ml of TNF, so that the susceptibility to TNF was low. An enhancing effect of TNF on the cytotoxic effect of almost all the chemotherapeutic agents tested was observed against VMRC-RCZ and A-498. Against VMRC-RCW and Caki-1, the combination of TNF with the chemotherapeutic agents did not result in an enhancing effect. Mitomycin C showed an enhancing effect of TNF against all four cell lines. These results suggested that the combined treatment of TNF with chemotherapeutic agents may have favorable results in clinical trials.     

生长抑素与肾癌的关系及肿瘤坏死因子的影响

为探索肾癌生物治疗的新途径，将肾癌细胞接种于裸鼠背部皮下，荷瘤裸鼠随机分为３个ＴＮＦ组及２个对照组。给药后第３天取血０５～０８ｍｌ，及移植瘤、瘤旁及正常组织各１ｇ，放射免疫法测定组织标本中生长抑素（ＳＳ）浓度。结果：肿瘤组织、对照组肿瘤旁、正常组织中ＳＳ浓度（ｐｇ／ｍｌ）依次为０．６７３、０９００、０５１４；ＴＮＦ组分别为０．７９１、０８７０、１０４３。结论：肾癌生长（对照组）时瘤旁组织中ＳＳ浓度最高，而肿瘤及正常组织中较低，提示瘤旁组织具有抑制肿瘤生长的作用。应用ＴＮＦ后ＳＳ含量以正常组织中最高，瘤旁及肿瘤组织较低。表明ＴＮＦ不但对肿瘤有直接杀伤作用，而且能促进机体增强抑制肿瘤生长、扩散作用。     

Studies on anti-tumor activity of tumor necrosis factor alpha against human renal cell carcinoma cells heterotransplanted into nude mice]

The anti-tumor activity of recombinant tumor necrosis factor alpha (rTNF alpha) against the renal cell carcinoma cell line KU-2 was studied in vivo, employing athymic nude mice. The nude mice were divided into six groups and each group was composed of six mice. Group I underwent no treatment, Groups II and III were injected with 5 and 10 micrograms of rTNF alpha intraperitoneally, respectively. Group IV received 5 micrograms of rTNF alpha intravenously and Groups V and VI were administered 2.5 and 5 micrograms of rTNF alpha six times, given intravenously every other day, respectively. Evaluation of the study was performed according to Battelle Colombus Laboratories Protocol. Tumor regression was defined as RW less than 1; inhibition of tumor proliferation was defined as TRW/CRW less than or equal to 42%. Other results were defined as no effect. No obvious anti-tumor activity was observed in group II and III. Though inhibition of tumor proliferation was noted in Group IV, death of two nude mice in this group was noted. When rTNF alpha was administered to the mice in Group V, complete disappearance of the heterotransplanted tumor in two nude mice and tumor regression in four were observed. No death of mice in this group occurred. When the dose of rTNF alpha was raised to 5 micrograms (Group VI), however, five mice died. The histopathological study revealed remarkable necrosis in the tumor tissue and congestion around the white pulps of the spleen 24 hours after intravenous administration of 5 micrograms of rTNF alpha, although no obvious changes were noted in the liver, kidney and digestive organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on in vitro anti-tumor activity of tumor necrosis factor alpha against human renal carcinoma cell line (KU-2)]

We attempted to clarify the anti-tumor activity and its mechanism of human recombinant tumor necrosis factor alpha (rTNF alpha). The established cell line KU-2, derived from human renal cell carcinoma, was treated with rTNF alpha alone or in combination with the following anti-cancer agents in vitro: actinomycin D (ACD), vinblastine sulfate (VLB), nimustine hydrochloride (ACNU), and methotrexate (MTX). In vitro studies, including cytotoxic assay, colony forming assay and flow cytometric DNA analysis were performed. By cytotoxic assay, 21.4 +/- 4.0% and 34.8 +/- 4.7% of the cells were killed by 72 hour incubation with 100 ng/ml of rTNF alpha alone, and 1 ng/ml of ACD alone, respectively. An augmented cytotoxicity of 75.3 +/- 0.3% was observed by simultaneously adding 1 ng/ml of rTNF alpha and 1 ng/ml ACD. However, when KU-2 was treated with both 100 ng/ml of rTNF alpha and 3 micrograms/ml of ACNU or 2.5 ng/ml of MTX, no significant increase in cytotoxicity was noted. In the colony forming assay study, the colony forming efficiency (CE) of the control cultures was 31.8 +/- 8.1%. A 92.3 +/- 1.8% reduction in CE was demonstrated when 100 ng/ml of rTNF alpha was added to the cultures. No augmented effects were seen between rTNF alpha and chemotherapeutic agents in this study. In flow cytometric DNA analysis, no cell cycle specific effects of rTNF alpha were demonstrated, regardless of whether or not chemotherapeutic agents were added. These results indicate that the cytotoxic and cytostatic activities of rTNF alpha may be mediated by separate mechanisms of action and that rTNF alpha affects more markedly KU-2 cells having clonogenic potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Anti-neoplastic activity of a human recombinant tumor necrosis factor (Hu-rec-TNF) alone or in combination with human recombinant gamma-interferon, doxorubicin or cis-platinum on a human renal cell carcinoma line

The inhibitory effect of human-recombinant tumor necrosis factor (Hu-rec-TNF) alone or in combination with human recombinant gamma-interferon (rec gamma-IFN), doxorubicin (ADM) or cis-platinum (CDDP) was studied for two human renal cell carcinoma lines (KO-RCC-1 and RCC-nu-1 cells). Hu-rec-TNF inhibited the cell growth of KO-RCC-1 cells in a dose- and time-dependent manner. The inhibitory effect was seen 48 hours after the treatment with Hu-rec-TNF alone. The effect of Hu-rec-TNF was cytotoxic in our experiment. On the other hand, Hu-rec-TNF did not inhibit the cell growth of RCC-nu-1 cells. There were sensitive and resistant cells in renal cell carcinoma. In KO-RCC-1 cells, Hu-rec-TNF enhanced the inhibitory effect of rec gamma-IFN, ADM or CDDP. On the other hand, Hu-rec-TNF did not enhance the inhibitory effect of rec gamma-IFN, ADM or CDDP in RCC-nu-1 cells. In our experiment, the combined treatment with Hu-rec-TNF and rec gamma-IFN, ADM or CDDP was useful in human renal cell carcinoma which was sensitive for Hu-rec-TNF.     

Inhibitory effects of tumor necrosis factor alpha on fracture healing in rats

Fracture healing, which involves a cascade of biological tissue responses, may be affected by various biochemical substances. One of these substances is tumor necrosis factor alpha (TNF). Studies were made on the effects of TNF on healing of fractured ribs of rats. Fracture healing was inhibited by daily administration of recombinant human TNF (400 μg/kg body weight per day, intraperitoneally) after fracture. The rate of union on day 20 was significantly lower in the TNF-treated group (4/18, 22.2%) than in the control group (14/18, 77.8%) (p < 0.001 by Chi-square test). Histological examination showed that TNF inhibited cartilagenous callus formation. On day 10, cartilage was seen in the gap zone and under the periosteum in the control group, but no cartilage formation was observed in the gap zone in 9 of 12 specimens from the TNF-treated group. On day 20, the fracture ends were united by newly formed bone in the control group, but mature fibrous tissue was seen in the gap zone, and bony or cartilagenous union was not achieved in the TNF-treated group. These results show that TNF inhibits cartilage formation in the early phase of bone induction in fracture healing and suggest that this effect of TNF is due to its inhibition of differentiation of mesenchymal cells into chondroblasts.     

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.     

肿瘤坏死因子与可溶性肿瘤坏死因子受体在膀胱肿瘤中的表达

目的探讨肿瘤坏死因子（TNF）和可溶性肿瘤坏死因子受体（STNFR）与膀胱肿瘤生物学行为的关系。方法测定26例膀优移行细胞癌患者和20例正常对照者血清中TNF和STNFR水平。结果发现膀胱肿瘤患者血TNF水平与对照组相比差异无显著性（P＞0．05）,而STNFR水平显著高于对照组（P＜0．001）,并随着临床分期的增加而升高,巨术后2周时显著下降（P＜001）;TNF与STNFR水平之间无显著相关关系。结论STNFR抑制了TNF生物学活性而降低了机体的免疫功能,并可作为评估病情严重程度和预后的指标。     

Evaluation of sequential plasma and urinary tumor necrosis factor alpha levels in renal allograft recipients

The macrophage cytokine tumor necrosis factor-alpha is released early in immune activation and may be detected in the peripheral circulation. This study has investigated the occurrence of plasma and urinary TNF in 30 renal allograft recipients. Although circulating TNF may be detected in 20% of pretransplant or normal control samples, levels were significantly elevated during 65% of allograft rejection episodes. Plasma TNF levels did not rise in graft failure due to acute tubular necrosis, but were always highly raised in systemic infection. In contrast, urinary TNF was only detected in association with acute rejection (49%) or tubular necrosis (14%), and no controls had detectable urinary TNF. These findings indicate that evaluation of circulating and excreted TNF may give further insight into the immunobiology of graft rejection.     

肿瘤坏死因子相关凋亡诱导配体受体在肾癌中的分布及其意义

目的：研究肿瘤坏死因子相关诱导凋亡配体（TRAIL）受体在肾癌组织中的分布及其意义。方法：采用RT－PCR及NorthernBlot的方法检测TRAIL受体在肾癌组织及正常肾组织,肾癌细胞系GRC－I和正常肾小管细胞系HK－2中的表达。结果：死亡受体DR4、DT4在肾癌组织及正常肾组织、肾癌细胞系GRC－I和正常肾小管细胞系HK－2中强表达;假受体DcR-1在正常肾组织和正常肾小管细胞系HK－2中强表达;假受体DcR-2未见表达。结论TRAIL基因在肾癌肿瘤细胞的凋亡机制中可能发挥重要的作用。     

Immunological effects of granulocyte-macrophage colony-stimulating factor and autologous tumor vaccine in patients with renal cell carcinoma

PURPOSE: Biological therapy for renal cell carcinoma (RCC) uses agents that mobilize immune effector cells which are able to recognize and destroy cancer. We evaluated the effects of weekly then monthly autologous tumor vaccine combined with daily granulocyte macrophage-colony stimulating factor (GM-CSF) in patients with RCC as a method of stimulating antigen presenting cells. MATERIALS AND METHODS: Eligible patients with pathological stage II to IV RCC were entered into this pilot study. Autologous tumor vaccine (0.5 to 1 x 107 irradiated tumor cells) admixed with 250 microg GM-CSF per vaccine was given subcutaneously weekly for 4 weeks and then monthly for 4 months. GM-CSF (125 microg/m2) was given subcutaneously for 13 days after vaccine injection 1 and injections 4 to 8. Treatment related tumor specific CD4 and CD8 positive T cell precursors were assessed. RESULTS: A total of 22 patients were entered into this study. Patients were stratified by bulk of disease (group 1, 9 patients with micrometastatic disease, and group 2, 13 patients with macrometastatic disease). In general treatment was well tolerated. Of 9 patients in group 1 7 remained disease-free after nephrectomy. In group 2, 6 patients had stable (46.2%) and 7 patients had progressive disease (53.8%). Statistically significant treatment related increases in CD4 (p = 0.028) and CD8 (p = 0.018) positive tumor specific T cell precursors were observed for the entire group of patients. Changes in CD4 and CD8 positive precursors correlated significantly with each other (p = 0.0001). This correlation was seen in the 2 patient subpopulations as well (group 1 p = 0.003, group 2 p = 0.013). Patients with minimal disease, and with changes in CD4 and CD8 positive tumor specific T cell precursors greater than the median appeared to have an improved time to progression as well as a survival benefit. CONCLUSIONS: GM-CSF and autologous vaccine can be given safely in combination to patients with renal cell cancer. We observed treatment related changes in tumor specific circulating lymphocyte populations.     

In vivo effects of diadenosine polyphosphates on rat renal microcirculation

BACKGROUND: Diadenosine polyphosphates (APXA) are vasoactive nucleotides that elicit effects via purinoceptors. Recent data suggest differential effects of APXA on kidney vasculature. METHODS: The in vivo effects of AP3A, AP5A, and adenosine on renal microvessels and the role of purinoceptors were investigated by the application of agonists to the hydronephrotic rat kidney and preincubation with respective antagonists. RESULTS: The addition of the agonists (10-7 mol/L up to 10-4 mol/L) resulted in a concentration-dependent transient vasoconstriction [interlobular artery (ILOB): adenosine 30 +/- 7%, N = 7, AP3A 35 +/- 10%, N = 5; AP5A 66 +/- 19%, N = 5; 10-5 mol/L each] lasting up to one minute, followed by a concentration-dependent vasodilation (ILOB: adenosine 10 +/- 3%, N = 6; AP3A 19 +/- 4%, N = 5; AP5A 12 +/- 5%, N = 6; 10-5 mol/L each). In ILOB and in the afferent arteriole (AFF), the constrictory effects of AP5A were more pronounced than those of AP3A and adenosine. In the efferent arteriole (EFF), vascular tone was only slightly affected by all agonists. The dilatory potency was comparable for all agonists in ILOB and EFF. No significant vasodilation occurred in AFF. The application of the selective A1 receptor antagonist DPCPX (10-5 mol/L) completely abolished the adenosine-induced vasoconstriction, whereas the A2 receptor antagonist DMPX and the P2 purinoceptor antagonists PPADS and A3P5P (all 10-5 mol/L) did not affect adenosine-induced constriction. The AP3A-induced constriction was abolished by DPCPX and was partially inhibited by PPADS. The constriction induced by AP5A was less sensitive to DPCPX but more sensitive to PPADS. In ILOB and EFF, DMPX or A3P5P abolished dilation after the addition of the agonists. The dilation after AP5A was not significantly reduced. In AFF, no significant dilation was observed with these agonists alone, but it was clearly visible in the presence of DPCPX or PPADS. CONCLUSIONS: APXA evoke transient constrictions in vessels of the hydronephrotic rat kidney, which are mediated by A1 and P2 purinoceptors. The length of the phosphate chain determines the degree of vasoconstriction and the extent to which the substances exert effects on the P2 purinoceptor subtypes. ILOB and AFF are more potently affected by APXA than EFF. Afferent vasodilation is partially overridden by sustained vasoconstriction.     

In vivo effects of tumor necrosis factor-a on capillary permeability and vascular tone in a skeletal muscle

Background. The present study aims at analysing short-term effects of TNF_α on capillary permeability, on transcapillary fluid fluxes, and on vascular tone in a whole organ cat skeletal muscle in vivo preparation.
Methods. The denervated muscle was isolated from the body but with intact vascular supply. The experimental setup allowed continuous recording of vascular tone, of hydrostatic capillary pressure and tissue volume variations. The capillary filtration coefficient (CFC), which represents the net effect for transcapillary fluid exchange of capillary permeability and number of open capillaries, was calculated before and during intra-arterial TNF infusion.
Results. We found that TNF had a minor effect on vascular tone in terms of a small vasodilation, and that no effect on hydrostatic capillary pressure could be recorded. CFC increased by 64% during the TNF infusion and this increase must be attributed to an increase in capillary permeability rather than an increase in the number of capillaries available for fluid exchange since the TNF effect on vascular tone is small. This was also supported by TNF-induced transcapillary filtration.
Conclusions. TNF is a potent drug for increasing capillary permeability causing transcapillary filtration in vivo. Its release, e.g. during sepsis, may therefore contribute to the capillary leakage often seen in this clinical situation.