In vitro antiproliferative effect of cancer chemotherapeutic agents and their combination with interferon in renal cell carcinoma

We studied an antiproliferative effect of cancer chemotherapeutic agents, interferon (IFN) and, in particular, their combination effect on renal cell carcinoma. Either of colony formation assay (CFA) or [3H]-thymidine incorporation assay ([3H]-TdR assay) was employed as an in vitro chemosensitivity testing system. When compared, these two systems produced similar results with a good correlation (r = 0.97, p less than 0.01), in the antiproliferative effect of 30 drugs for 4 primary renal cell carcinomas and 5 xenotransplantable renal cell carcinomas. In vitro chemosensitivity test (CFA or [3H]-TdR assay) screened successfully only 5 "sensitive" drugs (7.9%) out of a total 63 cancer chemotherapeutic agents for 24 human renal cell carcinoma. This confirmed the findings reported by others. In the study of the antiproliferative effect of a cancer chemotherapeutic agent, human lymphoblastoid interferon (HLBI) and their combination on human renal cel carcinoma cell line (SMK-R2). Each of VBL, MTX or HLBI tended to suppress [3H]-TdR uptake in a dose-dependent manner. The combination of VBL (0.05 microgram/ml) and HLBI (10(2) or 10(3) IU/ml) produced a subadditive effect, and that of MTX (0.1 microgram/ml) and HLBI 10(2) IU/ml produced a synergistic effect on the human renal cell carcinoma cell line, the effect which is evaluated by Valeriote and Lin's criteria of combination. In particular, the synergistic effect by MTX and HLBI under the clinically achievable drug concentration seems important, when its clinical application is considered.     

Effect of hyperthermia on the cytotoxicity of 4 chemotherapeutic agents currently used for the treatment of transitional cell carcinoma of the bladder: an in vitro study

PURPOSE: Hyperthermia combined with chemotherapy is not a novel cancer treatment. However, the working mechanism of this combination therapy is not fully understood. In the current in vitro study we investigated the differences in cytotoxicity of 4 chemotherapeutic agents at 37C or 43C. MATERIALS AND METHODS: The human transitional cell carcinoma cell lines used were RT4, RT112, 253J and T24. Cells were seeded in 96-well microtiter plates. After 24 hours cells were treated for 60 minutes with increasing concentrations of mitomycin C, epirubicin, gemcitabine and EO9 at a temperature of 37C or 43C. After treatment cells were rinsed 3 times and left for 24 hours in the incubator at 37C. The influence of chemotherapy and temperature on cell survival was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. RESULTS: Decreased cell proliferation with increasing concentrations of chemotherapeutic agents was demonstrated. EO9 proved to be the most potent agent at each temperature. Hyperthermia alone did not demonstrate decreased cell proliferation. However, a synergistic effect on decreased cell proliferation was demonstrated in all cell lines and chemotherapeutic agents used, although each had a maximum at a different chemotherapy concentration and to a different extent. Synergism was most obvious in cell lines treated with low dose epirubicin. CONCLUSIONS: Synergism with hyperthermia and chemotherapy was clearly demonstrated for epirubicin, EO9, mitomycin C and to a lesser extent gemcitabine. Hyperthermia alone did not cause decreased cell proliferation. Synergism was most prominent with low drug doses and the most potent drug used in this in vitro study was EO9.     

Growth inhibitory effect of p21 and p53 containing adenoviruses on transitional cell carcinoma cell lines in vitro and in vivo

Altered p53 expression has been demonstrated in the majority of advanced transitional cell carcinoma (TCC) of the bladder tumors. The objective of this investigation was to examine the effect of the introduction of a p53 or p21^(WAF1/CIP1) adenovirus on the proliferation and apoptosis of various human TCC cell lines in vitro and in vivo. Proliferation was measured by ³H-thymidine incorporation. Apoptosis was measured by DNA fragmentation and bax expression. We also examined the effect of ex vivo introduction of the p21^(WAF1/CIP1) or the p53 gene on growth of the T24 TCC cells and UMUC-3 TCC cells introduced subcutaneously into athymic nude mice. We found that although the effect of the p21-adenovirus on the proliferation of various TCC lines varied with each individual cell line, there was a substantial growth inhibition observed (greater than 80% growth inhibition) in seven of the eight TCC cell lines at the highest viral dosage. In contrast, after 24 h, the highest dosage of the p53-adenovirus produced only a heterogeneous decrease in proliferation compared to the highest dose of the p21^(WAF1/CIP1)-adenovirus (40–90%). In ex vivo experiments, no tumors were found in nude mice injected subcutaneously with either TCC cell line exposed in vitro to the AdSCMV-p21^(WAF1/CIP1) or AdSCMV-p53 viruses before three weeks. There was a threefold decrease in tumor square area at week 5 in the Ad5CMV-p21^(WAF1/CIP1) or Ad5CMV-p53 TCC cells injected mice (p<0.001, p<0.009) compared to either mock or Ad5CMVLacZ TCC bladder tumor cells. These data suggest that significant portion of the effect of altered p53 on TCC phenotype may be mediated through the p21^(WAF1/CIP1) pathway. Thus, the restoration of p21^(WAF1/CIP1) function in this tumor system may be a beneficial therapeutic strategy.     

Combined effect of interleukin 2 and cyclophosphamide in therapy of mice with transitional cell carcinoma

We investigated the effect of combination therapy with interleukin 2 (IL-2) and cyclophosphamide (CPM) in C3H/HeN mice implanted with mouse bladder tumor cells (MBT2). MBT2-bearing mice were treated with a single intraperitoneal injection of 120 mg/kg CPM on day 10 and/or subcutaneous administration of 5 x 10(4) units IL-2/day from day 11 to day 20. As a result, the growth of tumor in mice treated with IL-2 alone was slightly suppressed. On the other hand, regression was observed in all mice treated with CPM alone, although regrowth of tumor was seen in all of them. However, when IL-2 was administered with CPM, regrowth of tumor was completely suppressed. An immunologic study was done that showed cytotoxicity against YAC-1 and P815 in the spleen cells was suppressed in mice treated with CPM alone, but that both recovered to control levels when IL-2 was administered with CPM. Cytotoxicity against MBT2 in the spleen cells was most elevated in mice treated with both IL-2 and CPM. These findings suggested that clinical evaluation of combination therapy with IL-2 and CPM may be worthwhile.     

Interferon-alpha suppresses the antiapoptotic effect of NF-kB and sensitizes renal cell carcinoma cells in vitro to chemotherapeutic drugs

BACKGROUND: Immunochemotherapy (ICT) with interleukin-2 (IL-2) and interferon-alpha (IFNalpha) with a secondary effector (5-fluorouracil, 5 FU) is the only promising treatment for advanced renal cell carcinoma (RCC). With IFNalpha, besides the activation mechanisms of the immunosystem, a direct antitumor effect on tumor cells is expected. MATERIALS AND METHODS: NF-kB activity in three permanent cell lines (Hep2, HepG2, HT29) and in primary RCC cell lines was measured after incubation with tumor necrosis factor-alpha (TNFalpha), IFNalpha, IFN-gamma, TNFalpha+IFNalpha, and IFNgamma+TNFalpha, respectively. NF-kB activity and induction of apoptosis by chemotherapeutic drugs (5FU and doxorubicin) were determined in cells transfected with a constitutively active NF-kB p65 or a dominant negative IkB. RESULTS: NF-kB signaling induced by TNFalpha is suppressed by IFNalpha and IFNgamma in the permanent cell lines and in the primary RCC tumor cell cultures. In an in vitro ICT model we show that pretreatment of RCC with IL-2 and IFNalpha leads to a diminished NF-kB response to TNFalpha. In certain tumors, this correlates with increased susceptibility to investigated chemotherapeutic drugs as shown by annexin stain and cell elimination. Modulation of the cellular NF-kB state by a constitutively active p65 or a dominant negative IkB mimics this effect. The IkB construct leads to the same effects as IL-2/IFNalpha pretreatment as shown by predominant elimination of the transfected cells from the overall population, while introduction of p65 leads to a partial rescue from the effect of IL-2 and IFNalpha. The described effect, however, applies only to a selection of primary cell cultures. CONCLUSIONS: Besides the immunomodulation effects, treatment of RCC with IL-2/IFNalpha leads to a proapoptotic state in certain tumors. The relevant mediator seems to be IFNalpha by suppression of the antiapoptotic effect of NF-kB. These data can provide an experimental base for correlation with real patient outcome after ICT.     

Fluorescence in situ hybridization for detecting transitional cell carcinoma: implications for clinical practice

OBJECTIVE: To determine the diagnostic sensitivity of genetic studies using fluorescence in situ hybridization (FISH) for detecting both new and recurrent cases of transitional cell carcinoma (TCC) in a routine clinical practice setting, as bladder cancer has a significant risk of recurrence and progression to invasive disease and thus sensitive surveillance testing is very important. PATIENTS AND METHODS: FISH was performed using the UroVysion kit (Vysis Inc., Downers Grove, IL, USA) Consecutive patients were assessed using FISH, both to evaluate those with a history of TCC or with suspicious symptoms, and the FISH results were compared with concurrent biopsy and cytological assessments. RESULTS: In all, 521 consecutive FISH tests from 300 patients were evaluated; 47% had a history of bladder cancer and 53% had suspicious symptoms. Of the 521 FISH tests, 24% were positive; concurrent cytology was available for 84% of the FISH tests, with a concordance rate of 78% (6% were positive for both and 72% were negative by both tests). For the discordant cases, FISH was positive and cytology negative in 21% of cases, and cytology was positive with a negative FISH for 1%. In all, 99 FISH tests had concurrent biopsy data. Of the 44 cases histologically positive for TCC, 32 were FISH-positive, resulting in an overall sensitivity (95% confidence interval) of 73 (60-88)%. FISH detected 95% of cases with high-grade carcinoma, while only seven of these 17 were positive by concurrent cytological assessment. FISH detected 56% and cytology detected 32% of low-grade lesions. FISH detected all nine new cases with positive histology. Overall, the specificity of FISH was 65 (53-78)%. Of 112 patients with previous TCC, 28 had a recurrence; 22 of these had positive FISH results. CONCLUSION: FISH analysis has a high sensitivity for detecting new cases of TCC, as well as recurrences. From the present data FISH is considerably more sensitive and only slightly less specific than cytology in diagnosing TCC. Therefore, we recommend FISH as a useful initial diagnostic tool in patients suspected of both new and recurrent TCC.     

Effects of selected chemotherapeutic agents on PCNA expression in prostate carcinoma cell lines

Bivariate flow cytometric analysis of proliferating cell nuclear antigen (PCNA) was performed on prostate carcinoma cell lines (PC-3, DU-145). For both cell lines 100% methanol fixation provided optimal fluorescence intensity of PCNA. The ratio of PCNA/DNA increased in late G1 through early S/phase, followed by a decrease in mid- and late S and enhancement in G2/M phase. PCNA expression was increased in G2/M phase cells treated for 48 h with vinblastine. A slight decrease in PCNA expression was observed with cyclohexamide treatment. Hydroxyurea induced an increase in S-phase fraction along with enhanced PCNA expression. Methotrexate and Adriamycin had little effect on the cell cycle compartments of PC-3 or DU-145; however, methotrexate decreased PCNA expression, while Adriamycin enhanced it. Cisplatin increased S-phase in both cell lines, increasing PCNA expression in PC-3 and decreasing it in DU-145 cells. The data on the effects of drug treatment point to a dissociation between PCNA expression and S-phase fraction as calculated from the DNA distribution. In some cases, e.g., the cisplatin studies, different effects were obtained in the two different cell lines treated with the same drugs. Whether changes in PCNA expression will provide more useful information than S-phase fraction for evaluation of potential antitumor drugs is not known.Supported by NIH grants Ro-1-CA 14134     

Cinacalcet modifies the pH of solutions in vitro: possible implications for gastro-intestinal side effects in vivo.

Sir, The US-based Federal Drug Agency (FDA) and the European MedicinesAgency have approved the use of cinacalcet HCl for the treatmentof secondary hyperparathyroidism in patients on dialysis, andin those with parathyroid carcinoma [1]. Cinacalcet is effectiveand, overall, well tolerated [2]. However, in clinical trials,it has been observed that certain patients on cinacalcet haveepisodes of hypocalcaemia (1.4% of patients), nausea (31%) andvomiting (27%), much more than in patients receiving a placebo[3,4]. Gastrointestinal intolerance, with vomiting in particular,has been     

Antiepidermal growth factor receptor antibodies augment cytotoxicity of chemotherapeutic agents on squamous cell carcinoma cell lines.

Recent literature by Fan et al (1993) demonstrated that addition of cisplatin and monoclonal antibody to the epidermal growth factor receptor (EGFR MAb) of the human squamous cell carcinoma (SCC), A431, eradicated gross tumors in nude mice. To determine whether a combination of either cisplatin or 5-fluorouracil (5-FU) with EGFR MAb could affect an SCC other than the A431 tumor model, an assay using 2 human tongue SCC cell lines, BroTo and SCC-25, was performed. Cells were pretreated with 1.25 microgram/mL cisplatin or 10 microgram/mL 5-FU. After a 4-hour incubation period, cisplatin-treated cells were washed with phosphate-buffered saline solution. Various concentrations of EGFR MAb were then added, and after a 24-hour incubation period, an MTT cell growth assay was performed. SCC-25 cells exhibited a greater decrease in growth with the addition of 16 nmol/L EGFR MAb to cisplatin compared with the cytotoxicity of cisplatin alone (P < 0.001). However, this combination did not produce similar results with BroTo cells (P > 0.05). The combination of EGFR MAb and 5-FU produced a growth inhibition versus control (unexposed cells) in both cell lines (P < 0.05). These findings suggest the possible augmentation of the activity of chemotherapeutic agents, cisplatin and/or 5-FU, with the addition of EGFR MAb on SCC cell lines other than A431.     

Intravenous urography in patients with transitional cell carcinoma of the bladder. The incidence and implications of ureteral obstruction.

In order to study the value of excretory urography in the diagnosis of transitional cell carcinoma of the bladder, and also the incidence and implications of ureteral obstruction, 100 consecutive patients were studied. Of 73 patients with superficial tumours (stages Tis, Ta, T1) only 1 (1,4%) had hydronephrosis as a result of the bladder tumour. However, 2 further patients had hydronephrosis secondary to synchronous ureteral tumours. Of the 27 patients with muscle-invasive tumours, 10 (37%) had hydronephrosis at the time of diagnosis. Four patients who had normal upper tracts initially, developed hydronephrosis during follow-up: 1 due to progression of a superficial tumour to stage T3, 1 due to the development of an ureteral tumour, and 2 due to fibrosis of the intramural ureter after transurethral resection of superficial tumours. The presence of ureteral obstruction at the time of diagnosis most often implies a muscle-invasive tumour, but the possibility of a synchronous ureteral tumour must also be considered. Fibrous strictures of the distal ureter can occur after transurethral resection of superficial bladder tumours.     

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.     

In vitro study of the effect of hyperthermia on normal bladder cell line and on five different transitional cell carcinoma cell lines.

Intraluminal hyperthermia is potentially useful in the management of superficial bladder cancer. The potential inhibitory effect of hyperthermia on various human bladder cancer cell lines, normal human bladder cells and the murine MBT-2 bladder cancer cell line has been studied in vitro. These cell lines were exposed for one hour to 43 +/- 0.5C and compared to controls. Cell survival was assessed comparing the cell growth curve and colony formation. The human transitional cell carcinoma (TCC) cell lines vary in their sensitivity to heat. MGH-U1 was the most heat sensitive cell line. The human A-1698, CUB-2, UM-UC-3 and the murine MBT-2 lines were heat insensitive. We conclude that the cytocidal effect of hyperthermia in bladder transitional cell carcinoma is variable. Further experiments using the combination of hyperthermia and intravesical anticancer agents are in progress.     

Technique for in vivo perfusion of chemotherapeutic agents into the rat liver.

The toxic side effects of many cancer chemotherapeutic agents prevent their use in high concentrations. This paper describes a technique for the administration of large doses of chemotherapeutic agents to the rat liver in an in vivo isolated perfusion system. This method is applicable for testing the effect of various agents on liver pathophysiology. No alteration in liver function, liver weight, or histological pattern was noted following perfusion with high concentrations of 5-fluorouracil.     

特异性环氧合酶-2抑制剂与不同抗癌药联用对胶质瘤细胞生长的影响

目的 观察特异性环氧合酶-2抑制剂依托昔布和塞来昔布(celebrex)与长春新碱(VCR)、顺铂(DDP)、鬼臼噻吩甙(VM-26)等抗癌药联用对胶质瘤细胞增殖的影响.方法 30 μmol/L依托昔布、100 μmol/L塞来昔布与不同浓度的VCR、DDP、VM-26单用、联用48 h.噻唑蓝(MTT)比色法检测各组抑制率,中效原理和金正均法评价特异性环氧合酶-2抑制剂与各抗癌药联用的效果.结果 30μmol/L依托昔布和100 μmol/L塞来昔布单用对胶质瘤细胞增殖抑制率分别为(40.87±1.88)%和(46.81±2.37)%;不同浓度(1、10、100 mg/L)的VCR、DDP、VM-26单用对胶质瘤细胞增殖均有不同程度的抑制作用;依托昔布和塞来昔布与不同浓度VCR、DDP、VM-26联用组的抑制率均高于各药物单用组,具有协同作用.结论 依托昔布和塞来昔布与抗癌药联用后起协同作用,特异性环氧合酶-2抑制剂可作为抗癌药的化疗增敏剂.     

Human pancreatic carcinoma cells are sensitive to photodynamic therapy in vitro and in vivo.

BACKGROUND: The aim of this study was to assess the efficiency of photodynamic therapy (PDT) on human pancreatic cancer cells in vitro and in an animal model. METHODS: Human pancreatic tumour cell lines were submitted to PDT with pheophorbide a (Ph a), a chlorophyll derivative, in culture and after grafting into athymic mice. Ph a was tested in culture (10-10-10-5 mol/l) with a 5-J/cm2 energy treatment and on tumour-bearing Nude mice (30 mg/kg intraperitoneally) with a 100-J/cm2 PDT session. The effect of PDT was assessed in vitro using proliferative, apoptotic and clonogenic tests and in vivo on tumour growth and on the induction of tumour necrosis. RESULTS: PDT inhibited tumour cell growth in culture by affecting DNA integrity. This tumour cell photodamage started at low concentration (10-7 mol/l) as corroborated by clonogenic and tumour growth tests. A strong necrosis was achieved in vivo with a single PDT session. CONCLUSION: PDT destroyed human pancreatic carcinoma after low photosensitizer supply and weak energy application. It exerted this tumoricidal effect via apoptosis induction with a gentle protocol, and apoptosis and/or necrosis with a stronger protocol.     

The synergistic effect of hyperthermia and chemotherapy on murine transitional cell carcinoma

The in vivo effect of hyperthermia and chemotherapy was studied in a murine transitional cell carcinoma model. Localized hyperthermia (43.5C) of 60 and 90 minutes duration was combined with systemic doxorubicin hydrochloride, cis-platinum, cyclophosphamide or mitomycin to treat tumors implanted into the hind legs of C3H mice. The data were compared to the results obtained from the application of hyperthermia or chemotherapy alone as well as to the natural growth rate of untreated tumors. Untreated tumors grew with an exponential rate and had a doubling time of 4 +/- 1.5 days. Animals bearing such tumors survived for 25 +/- 7 days. When treated with hyperthermia alone, there was no significant reduction in the growth rate and no improvement was noted in the survival time. Treatment with doxorubicin hydrochloride, cyclophosphamide or mitomycin administered alone was likewise not effective. Cis-platinum alone was able to induce a minimal decrease in the growth rate. When the administration of chemotherapy was accompanied by hyperthermia, significant synergistic effect was noted for doxorubicin hydrochloride, cis-platinum and cyclophosphamide (p less than .01); only the mitomycin and hyperthermia combination failed to improve survival and decrease the growth rate. The duration of the hyperthermia exposure influenced the degree of tumor response. Hyperthermia of 90 minutes duration resulted in consistently greater decrease in tumor growth rate with doxorubicin hydrochloride, cis-platinum or cyclophosphamide than 60 minutes of hyperthermia combined with the same agents. These results indicate that local hyperthermia combined with doxorubicin hydrochloride, cis-platinum or cyclophosphamide can induce tumor regression, increase tumor doubling time and improve the survival of the tumor-bearing animal. Only the hyperthermia-mitomycin combination did not result in significant improvement from the baseline values. Thus, hyperthermia combined with selected chemotherapeutic agents can have an adjuvant effect in the treatment of established, implanted mouse bladder tumors.     

Combined effect of interleukin 2 and bacillus Calmette-Guérin in the therapy of mice with transitional cell carcinoma.

We investigated the effect of combination therapy with interleukin 2 (IL-2) and Bacillus Calmette-Guérin (BCG) on C3H/HeN mice implanted with mouse bladder tumor cells (MBT2). MBT2-bearing mice were treated with a local injection of BCG into the tumor at a dose of 1 mg/day for a total of 3 times and/or a 10-day continuous subcutaneous infusion of 5 x 10(4) units IL-2/day from day 11 to day 20. As a result, the growth of the tumor in mice treated with IL-2 alone was slightly suppressed, while tumor growth was hardly suppressed in mice treated with BCG alone. However, when IL-2 was administered with BCG, tumor growth was strongly suppressed and mean survival time was prolonged. Natural killer cell activity in the spleen cells was most elevated in mice treated with IL-2 and BCG, while lymphokine-activated killer cell activity was not observed in all groups. Lymphocyte subset analysis showed that there was little change in the ratio of Lyt2-positive lymphocytes or that of L3T4-positive lymphocytes. These findings suggested that clinical evaluation of combination therapy with IL-2 and BCG may be worthwhile.