Chemotherapeutic efficacy of the protein-doxorubicin conjugates on multidrug resistant rat hepatoma cell line in vitro.

In vitro studies were initiated to study the antitumour effect of protein-doxorubicin (DXR) conjugate on the growth of the multidrug resistant rat ascites hepatoma cell line, AH66DR. The 50% inhibitory concentration (IC50) for DXR in AH66DR cell line was 16 mumol l-1 (AH66 parental cell line, AH66P, IC50 was 0.08 mumol l-1). Treatment of AH66P and AH66DR cells with various concentrations of DXR or conjugates at equivalent concentrations of DXR was performed. The two types of conjugates used were bovine serum albumin (BSA)-DXR conjugate and immunoglobulin G (IgG)-DXR conjugate. Both of these conjugates showed potent dose-dependent inhibition of cell growth against AH66DR cells as compared with the cells treated with DXR or other controls. The IC50 for BSA-DXR and IgG-DXR conjugates in AH66DR cell line was 0.05 (equivalent DXR) mumol l-1 and 0.07 (equivalent DXR) mumol l-1, respectively. These values were similar to that of the AH66P treated with DXR. Cellular uptake and accumulation of DXR or BSA-DXR conjugate was also quantitated in both cell lines. The cellular concentration of DXR in AH66DR cells was 2-fold lower than that of AH66P cells throughout the experiment. In contrast, by the treatment of AH66DR cells with BSA-DXR conjugate, the intracellular drug concentration increased as a function of time up to 24 h (639.1 +/- 41.8, equivalent DXR, ng 10(-5) cells) and reached the same drug level as AH66P cells treated with DXR (617.9 +/- 17.3 ng-5 cells). Ammonium chloride treatment inhibited the effects of the conjugates but did not inhibit the free drugs. Intracellular DXR was effluxed rapidly from AH66DR cells, but BSA-DXR conjugate remained in the cells at relatively high concentration for a long time. These results indicate that by chemically modifying DXR, such as by conjugation of the drug with proteins, it may be possible to overcome multidrug resistance.     

In vitro and in vivo antitumor activity of lymphokine-induced cytotoxic cells

The present study demonstrates that LICC possess both in vitro and in vivo antitumor activity. The LICC were generated by culturing normal spleen cells with syngeneic peritoneal cells and indomethacin, or with a conditioned medium containing IL 2 with or without a putative new lymphokine, the CCDF. The LICC thus generated selectively killed the lymphoid or solid tumor targets of different H-2 haplotypes and of different etiological origins. The precursors of LICC were probably NK-like cells. The effectors were neither classical NK nor classical cytotoxic T lymphocytes. The LICC were very effective in preventing growth of both lymphoid and solid tumors in vivo, and Thy I+ cells were essential for the anti-tumor effect. The ability to generate LICC was preserved in the tumor-bearing hosts until the terminal stage of tumor growth, when the generation of suppressor T-cells interfered with LICC induction. LICC seem to play an important role in defense against non-immunogenic tumors.     

In vitro antitumor activity of silybin nanosuspension in PC-3 cells

The present study aims to evaluate the antitumor activity of silybin nanosuspension on human prostatic carcinoma PC-3 cell line in vitro. Silybin nanosuspension was prepared by the high pressure homogenization (HPH) method. MTT assay, observation of morphological changes and apoptotic body showed that silybin nanosuspension could significantly enhance the in vitro cytotoxicity against PC-3 cells compared to the silybin solution. Flow cytometric (FCM) analysis demonstrated that silybin nanosuspension induced G1 cycle arrest and apoptosis in PC-3 cells. Thereby, the overall results suggest that the silybin nanosuspension represents a potential source of medicine for the treatment of human prostate cancer.     

In vitro antitumor activity of tumor-infiltrating lymphocytes from human gastric carcinoma

Tumor-infiltrating lymphocytes (TIL) isolated from 11 gastric carcinoma were studied. TIL could grow for a long-term in medium containing recombinant interleukin-2(rIL-2). The mean expansion fold achieved in 6 long-term cultures of 11 specimens was 15.1 RIL-2 expanded gastric TIL exhibited significant cytotoxicity against K562, BGC823, MCF-7 and more effective antitumor cytotoxicity against fresh autologous tumor targets and human gastric cancer cell line. Peak cytotoxicity was shown in the third or fourth week after cultures. Cryopreservation of gastric TIL didn’t influence their expansion capacity and antitumor activity. Phenotypic analysis was demonstrated in this study. The results of present study indicate that TIL from human gastric carcinoma could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL-2. Their function may be of clinical importance.     

In vitro antitumor activity of bropirimine against urinary bladder tumor cells

BACKGROUND: Bropirimine is an orally-active immunostimulant that has an antitumor effect on superficial transitional cell carcinoma of the bladder. The mechanism of its antitumor effect has not yet been clarified. MATERIALS AND METHODS: The cytokine-mediated antiproliferative activity of bropirimine was tested against cultured KK-47 and 724 cells using a modified human clonogenic tumor assay. Perpheral blood mononuclear cells (PBMCs) obtained from five healthy volunteers were co-cultured with bropirimine and the levels of various cytokines, including IFN-alpha, gamma, IL-1beta and TNF-alpha in the supernatants, were quantified. RESULTS: Colony formation by both cell lines was directly inhibited by bropirimine in a dose-dependent manner. In co-cultures of bropirimine and PBMCs, the inhibition of colony formation by both cell lines was not enhanced compared with the effect of bropirimine alone. No significant elevation of cytokines was detected in the presence of PBMCs. CONCLUSION: The results obtained suggest that bropirimine is like to have direct antitumor activity rather than a cytokine-mediated antitumor effect.     

精子蛋白17单克隆抗体-多柔比星免疫偶联物体内外靶向抗卵巢癌活性研究

目的:将抗精子蛋白17(anti-Sp17)单克隆抗体(3C12)与多柔比星(DOX)偶联,并考察偶联物的体内外抗卵巢癌作用.方法:以二琥珀酰亚胺基酒石酸盐(DST)为交联剂制备3C12-DOX偶联物;免疫荧光法检测3C12-DOX的内化作用;MTT法测定3C12-DOX对Sp17+卵巢癌细胞的杀伤作用,流式细胞仪检测细胞的凋亡率.建立高表达Sp17荷瘤裸鼠模型,分为5组给药,分别经尾静脉注入生理盐水、3C12、S2D11-DOX、3C12-DOX和DOX,定期测量肿瘤体积和裸鼠体质量;实验结束时检测血液AST、LDH和CK-MB,检测心肌组织MDA和SOD,比较3C12-DOX和DOX对心脏的毒性.各组肿瘤称取质量,并检测肿瘤组织中细胞的凋亡率.结果:Sp17单克隆抗体(3C12)与DOX成功偶联,形成的3C12-DOX偶联物能够与Sp17+的细胞结合,并内化至细胞内;MTT结果显示,3C12-DOX偶联物能明显杀伤SK-OV3和HO8910细胞,加速细胞凋亡.在体实验中,注射3C12-DOX偶联物组的肿瘤体积明显小于其他对照组(DOX组除外).而与DOX组对比,3C12-DOX毒副作用明显降低.结论:3C12-DOX是潜在的靶向治疗卵巢癌的药物.     

In vivo antitumor activity of ilmofosine

Ilmofosine is a cytostatic/cytotoxic thioether phospholipid derivative. The in vivo anti-tumour activity of this compound was investigated in a methylcholanthrene (MethA)-induced fibrosarcoma and in the 3Lewis-lung carcinoma systems, respectively. Ilmofosine showed antineoplastic and antimetastatic properties at oral doses ranging from 0.625 to 40 mg/kg/day. Combination of Ilmofosine (p.o.) together with either cyclophosphamide (p.o.) or cis-DDP (i.v.) resulted in synergistic effects in vivo. These results demonstrate the in vivo antitumour activity of Ilmofosine in two tumour systems. The data indicate that direct cytostatic/cytotoxic effects of Ilmofosine are mainly responsible for its antitumour activity in vivo and which are increased by other cytotoxics.     

In vivo and in vitro antitumor activity expressed by cells of concomitantly immune mice

Growth of a primary tumor is often accompanied by the development of resistance to subsequent challenge implants of the same tumor, i.e., concomitant immunity. Using the P815 mastocytoma tumor, the kinetics of concomitant immunity was found to be governed by duration of exposure to the tumor and tumor mass. By implanting small "challenges" prior to the immunizing tumor, resistance to the growth of existing tumor foci was demonstrated. Winn-type assays revealed that antitumor activity was present in cell populations from the peritoneal exudate and lymph node draining the tumor. Peritoneal exudate cells, when infused systemically, were also able to confer protection against P815 mastocytoma challenge, suggesting their role as mediators of concomitant immunity. The 51Cr release technique indicated that cytolytic activity in lymph node cells, peritoneal exudate cells, and the spleen was present over a time course parallel to the kinetics of in vivo challenge. The peritoneal resident cell population was only slightly active; thus, effectors accumulated in the inflammatory exudate. Removal of specific subsets of cells from effector populations with antibody to surface markers and complement produced similar effects on both Winn and cytolytic assays. Anti-Thy 1.2 ablated measurable activity. It was substantially but not completely reduced by anti-Lyt 1.1 and only to a small degree by anti-Lyt 2.1.     

In vitro and in vivo antitumor activity of liposomal Fenretinide targeted to human neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid Fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). We investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-SIL showed specific, competitive binding to and uptake by, various NB cell lines. In in vitro cytotoxicity studies, NB cells, incubated with 30 microM HPR entrapped in anti-GD2-immunoliposomes, showed a significant reduction in cellular growth compared to free HPR, HPR entrapped in Ab-free liposomes or anti-GD2 empty liposomes. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. Similar, but significantly less potent, antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 MAb alone (p = 0.0297 and p = 0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that although anti-GD2 MAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only after treatment with anti-GD2-SIL-HPR (p < 0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.     

In vivo antitumor activity of tumor-infiltrating lymphocytes expanded in recombinant interleukin-2

A method was described for the generation of cells from tumor-bearing mice; these cells were capable of exhibiting significant antitumor reactivity when adoptively transferred into tumor-bearing hosts. Tumor cell suspensions from a variety of tumors were able to be separated using enzymatic techniques and they were cultured in medium containing recombinant interleukin-2. Activated infiltrating lymphocytes within these tumors expanded; and, by 6-8 days after initiation of culture, lymphocytes predominated and were able to grow to large numbers. The adoptive transfer of these tumor-infiltrating lymphocytes (TILs) made possible mediation of the reduction of established 3-day pulmonary micrometastases from 5 of 7 tumors tested, including two 3-methylcholanthrene (CAS: 56-49-5)-induced sarcomas, one 1,2-dimethylhydrazine (CAS: 540-73-8)-induced colon carcinoma, and the B16 melanoma, all in C57BL/6 mice, as well as the 1660 bladder carcinoma in BALB/c mice. Approximately 2-4 X 10(6) transferred cells were capable of totally eliminating 3-day established metastases. These cells were thus 50 to 100 times more effective than lymphokine-activated killer cells in reducing established metastases; however, they could not be generated from all tumors. The concomitant administration of recombinant interleukin-2 enhanced, by approximately fivefold, the in vivo activity of these cells. The specificity of action of TILs in vivo was different from that determined by classic amputation rechallenge experiments. The tumor-infiltrating lymphocytes that developed this antitumor reactivity appeared to be Thy-1+ and did not bear the asialo GM1 antigen. The potent antitumor effect of these TILs, when transferred in vivo to tumor-bearing hosts, raises the possibility of utilizing similar approaches for the isolation and therapeutic use of lymphocytes with antitumor reactivity from human tumors.     

In vitro and in vivo antitumor effect of 2-methoxyestradiol on human melanoma

2-methoxyestradiol (2ME(2)) is an endogenous metabolite of estradiol with estrogen-receptor-independent antitumor and antiangiogenic activity. We examined the effects of 2ME(2) on the cellular proliferation of 8 human melanoma cell lines. We show that 2ME(2) inhibited cell proliferation by inducing apoptosis and an arrest in the G(2)/M phase, and the mechanism of action involved microtubules, mitochondrial damage and caspase activation. In male SCID mice, 2ME(2) was effective in reducing primary tumor weight and the number of liver metastases after intrasplenic injection of human melanoma cells. In the metastases, we found a significantly higher rate of apoptotic cells after 2ME(2) treatment. These findings on the antitumor effect of 2ME(2) in cell culture as well as in an animal model may have implications for designing alternative treatment options for patients with advanced malignant melanoma.     

Daunomycin-immunoglobulin conjugates,uptake and activity in vitro

Daunomycin, an anti-tumor drug, the activity of which is known to be exerted mostly in the nucleus, was covalently attached to anti-tumor antibodies or to normal immunoglobulin. The attachment was accomplished either by direct linking or via a dextran bridge. All the conjugates retained a significant amount of their original drug activity. The capacity of these macromolecular conjugates to penetrate the malignant cell and the nucleus was examined. The compounds were found to be able to penetrate the cell membrane in the conjugated form. Daunomycin linked to anti-tumor antibodies entered the cell at a higher rate than daunomycin attached to normal Ig and daunomycin-dextran. In normal cells, no difference in uptake was observed between drug-immunoglobulin or drug-anti-tumor-antibody conjugates. Uptake studies with daunomycin conjugates, radioactively labeled by different methods, suggest that the drug is released from the complex after penetration into the cell but probably enters the nucleus as the free drug.     

In vivo antitumor activity of Sindbis viral vectors

BACKGROUND: Sindbis virus, a blood-borne virus transmitted by mosquitoes, has been used as a vector to efficiently express exogenous genes in vitro and in vivo and to induce apoptosis. Because Sindbis virus infects mammalian cells by interacting with the high-affinity laminin receptors, which are expressed at higher levels in several human cancers than in normal cells, we determined whether a Sindbis viral vector could be used to target cancers in vivo. METHODS: C.B-17-SCID mice with established xenografts were given daily intraperitoneal injections of the Sindbis viral vector SinRep/LacZ containing the bacterial beta-galactosidase gene. Control mice were untreated or received injections with phosphate-buffered saline. Tumor size was measured daily. Expression of beta-galactosidase and Factor VIII (a marker for endothelial cells) was determined by immunohistochemical staining of tumor sections. Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining. C.B-17-SCID beige mice, which lack natural killer (NK) cells, were used to assess the importance of NK cells in antitumor efficacy of Sindbis viral vectors. RESULTS: Tumors from mice treated with SinRep/LacZ were statistically significantly smaller than tumors from control mice. This effect was observed for tumor xenografts derived from BHK (kidney, hamster), LS174T (colon, human), HT29 (colon, human), and CFPAC (pancreas, human) cells. Expression of beta-galactosidase co-localized with that of Factor VIII in tumor sections. Tumors from SinRep/LacZ-treated mice contained more apoptotic cells than tumors from control mice. Complete tumor regression was observed in three of five C.B-17-SCID mice but in none of five C.B-17-SCID beige mice treated with SinRep/LacZ. CONCLUSION: Sindbis viral vectors efficiently targeted tumors in vivo, were apparently delivered through the circulation, and were more effective in the presence of NK cells.     

In vitro antitumor activity of teniposide against carcinoma of the lung in human tumor clonogenic assay

The antitumor activity of teniposide (VM26) on carcinoma of the lung was tested by comparing it with its congener, etoposide (VP16) in an in vitro soft agar clonogenic assay system (HTCA). Of 56 tumor biopsies placed in culture, 40 tumors were evaluable for drug sensitivity information (i.e., greater than or equal to 30 colonies in the control plate). The overall in vitro response rate (defined as less than or equal to 50% survival of TCFU) at the standard dose (10% peak plasma concentration) was 35%, which is higher than that of VP16 (28%). In cell lines derived from human carcinoma of the lung, VM26 showed 50% inhibition against colony formation of PC-7 cells at 0.45 microgram/ml which is less than that of VP16. A superior antitumor activity of VM26 on PC-9 cells was also observed. VM26 was observed to act faster than VP16 thus indicating its possible superior antitumor activity in vivo.     

In vitro activity (MIC and MFC) of voriconazole, amphotericin B, and itraconazole against 192 filamentous fungi: the GISIA-2 study

We evaluated the in vitro activity of voriconazole, amphotericin B, and itraconazole against 192 clinical mould isolates recovered in twenty Italian microbiology laboratories. The vast majority of isolates belonged to the genus Aspergillus (94.2%) with A. fumigatus (58.3%) being the most frequently isolated species. Antifungal susceptibility testing was performed using the broth microdilution method defined by the CLSI M38-A standard, and results were compared to those obtained with Sensititre panels. Aspergillus flavus ATCC 204304 was employed as reference strain and results were within all expected ranges. Voriconazole's activity against the 192 mould isolates was comparable to that of amphotericin B and itraconazole: voriconazole MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml), itraconazole MIC90 (CLSI 0.5 microg/ml, Sensititre 0.5 microg/ml), amphotericin B MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml). In conclusion, these in vitro data highlight voriconazole's broad spectrum activity against filamentous fungi and support its use as a first line agent for the treatment of fungal diseases.     

The in vitro and in vivo anti-tumour activity of N-AcMEL-(Fab')2 conjugates

To increase the accessibility of drug-antibody complexes to tumours and to decrease non-specific binding via Fc receptors N-acetyl-melphalan (N-AcMEL) was conjugated to F(ab')2 fragments. These fragments were synthesised by pepsin degradation of IgG MoAb. Up to 20 molecules of N-AcMEL could be successfully coupled to each F(ab')2 fragment (compared with 25 molecules/intact IgG) with retention of both drug and antibody activity. The N-AcMEL-F(ab')2 conjugates demonstrated specific cytotoxicity in vitro however despite the absence of non specific Fc receptor binding and greater permeability when using F(ab')2 fragments, the N-AcMEL-F(ab')2 and N-AcMEL-IgG conjugates had similar anti-tumour activity in vivo. Conjugates made with whole IgG and F(ab')2 were equally effective in eradicating subcutaneous solid tumours in mice when injected intravenously. The lower immunogenicity of F(ab')2 fragments compared with whole IgG and the similar cytotoxicity of their conjugates, suggests that the F(ab')2 conjugate has greater clinical utility.     

In vivo antitumor activity of S 16020-2, a new olivacine derivative

 The antitumor activity of S 16020-2, a new olivacine derivative, was investigated in vivo and compared with that of Adriamycin and elliptinium acetate in a panel of murine (P388 leukemia, M5076 sarcoma, Lewis lung carcinoma, and B16 melanoma) and human (NCI-H460 non-small-cell lung and MCF7 breast carcinomas) tumor models. S 16020-2 given i.v. was active against P388 leukemia implanted i.p., s.c., or intracerebrally. The therapeutic effect of an intermittent schedule (administration on days 1, 5, 9) was superior to that of single-dose treatment, allowing the i.v. administration of high total doses of S 16020-2 and resulting in the cure of 60% of mice in the i.p. P388 model. In this model, S 16020-2 was more active than elliptinium acetate and showed a better therapeutic index than Adriamycin:≥8 versus 2. A good therapeutic effect of S 16020-2 was also observed in three P388 leukemia sublines displaying the classic multidrug-resistance phenotype, namely, P388/VCR, P388/VCR-20, and P388/MDRC.04, the latter being totally insensitive to vincristine and Adriamycin. However, S 16020-2 was not active against the P388/ADR leukemia, a model highly resistant to adriamycin in vivo. S 16020-2 was both more active than Adriamycin and curative in the M5076 sarcoma and Lewis lung carcinoma implanted s.c. In the B16 melanoma implanted i.p. or s.c., S 160202 was less active than Adriamycin. Against the NCI-H460 human tumor xenograft, S 16020-2 demonstrated activity superior to that of Adriamycin (T/C=20% versus 43% on day 21). Against the MCF7 breast cancer xenograft, S 16020-2 was active, but less so than Adriamycin (T/C=23% versus 9% on day 21), whereas elliptinium acetate was marginally active (T/C=49% on day 24). The hematological toxicity of S 16020-2 given to B6D2F1 mice at pharmacological dose appeared to be less severe than that of Adriamycin, particularly in bone-marrow stem cells. These results demonstrate that S 16020-2 is a highly active antitumor drug in various experimental tumor models and is markedly more efficient than elliptinium acetate. Because of its pharmacological profile, which is globally different from that of Adriamycin, S 16020-2 is considered an interesting candidate for clinical trials. Received: 21 October 1995/Accepted: 4 March 1996     

In vitro and in vivo antitumor activity of the novel trinuclear platinum complex BBR 3464 in neuroblastoma

PURPOSE: BBR 3464 is a promising new trinuclear platinum complex that has been shown to circumvent the resistance to cisplatin in a panel of tumor cell lines and xenografts with acquired or intrinsic resistance to cisplatin. The in vitro and in vivo antitumor activity of BBR 3464 was evaluated and compared with that of cisplatin in neuroblastoma. METHODS: In in vitro studies, the short- and long-term cytotoxicities, cell cycle perturbations, the ability to induce apoptosis, the intracellular platinum accumulation and DNA platination were evaluated in three neuroblastoma cell lines exposed to appropriate drug concentrations for 1 h. In in vivo studies, BBR 3464 was administered i.v. at doses of 0.30 and 0.35 mg/kg three times at intervals of 4 days (q4dx3), and cisplatin was administered i.v. according to two different schedules (at 2 and 4 mg/kg three times at intervals of 4 days and at 6 and 12 mg/kg as single doses). RESULTS: In a short-term growth inhibition assay, BBR 3464 was shown to be up to 100-fold more potent than cisplatin and it was even more potent in a clonogenic assay. The difference in the antitumor effect of BBR 3464 on the different cell lines was evident in both assays, while cisplatin exerted a comparable antitumor activity in all lines tested. Cell cycle analysis demonstrated a longer-lasting block in G2/M phase induced by BBR 3464 without the early S phase accumulation induced by cisplatin. The higher potency of BBR 3464 appeared to be unrelated to the induction of apoptosis, that was lower or at most comparable to cisplatin. Cellular platinum accumulation and platinum-DNA adduct formation following BBR 3464 exposure was higher than following cisplatin exposure. These differences may have resulted from a different mechanism of action and may explain the lack of cross-resistance with cisplatin. In xenografts of neuroblastoma, BBR 3464 was confirmed to be very potent as compared to cisplatin (MTD 0.35 mg/kg and 4 mg/kg for BBR 3464 and cisplatin, respectively). The efficacy of BBR 3464 was superior to that of cisplatin when both drugs were administered on a fractionated schedule (q4dx3), while BBR 3464 appeared equally active to 12 mg/kg cisplatin administered as a single dose. CONCLUSIONS: Our findings indicate that BBR 3464 has a definite antitumor effect in neuroblastoma lines and may be a candidate for early clinical trials in children with neuroblastoma.