Antineoplastic activity of idazoxan hydrochloride.

PURPOSE: Idazoxan hydrochloride (IDA) is a 241 molecular weight imidazoline and adrenoreceptor ligand. It binds to mitochondrial membranes and promotes apoptosis of pancreatic beta cells. Since IDA has not been tested against tumor cells, the purpose of our study was to determine if IDA has antineoplastic activity. METHODS: We used the conversion of a soluble tetrazolium salt to an insoluble formazan precipitate and differential staining cytotoxicity assays to determine if IDA was cytotoxic to cell lines of murine lung cancer and human prostate cancer, as well as to a variety of fresh human tumor samples. We used flow cytometry to analyze cell death and calreticulin expression. RESULTS: IDA is cytotoxic to both cell lines and against aliquots of specimens of breast, gastric, lung, ovarian and prostate cancers as well as non-Hodgkin's lymphoma. It produces apoptotic cell death and promotes calreticulin expression, suggesting that IDA might be immunomodulatory in vivo. CONCLUSION: We anticipate that IDA will be clinically useful in cancer treatment.

Etoposide induction of tumor immunity in Lewis lung cancer.

PURPOSE: To determine if the antineoplastic effect of etoposide includes alteration in Lewis lung cancer cells which evoke an immunologic response in C57B1/6 host mice. METHODS AND RESULTS: Of C57B1/6 mice injected with 10(6) Lewis lung cancer (3LL) cells followed by treatment with a single 50 mg/kg dose of etoposide (VP-16), 60% survived over 60 days, in contrast to untreated control mice which died within 30 days. Approximately 40% of surviving mice rejected a subsequent challenge with 3LL. Their splenocytes protected naive mice injected with 3LL. To test if VP-16 treatment produced alterations in 3LL cells, which induce host immunity, leading to tumor rejection, C57B1/6 mice were injected with 3LL cells that had survived an 80-90% lethal concentration of VP-16 in vitro. These cells killed 75% of recipient mice but 60% of the surviving mice rejected challenge with 3LL. Splenocytes harvested from tumor-rejecting mice protected naive mice injected with 3LL. CONCLUSION: These results support the hypothesis that in addition to its antineoplastic cytotoxic effect, VP-16 induces changes in 3LL cells which are recognized by the host immune system resulting in immune rejection of 3LL. often immunosuppressive and therapeutic advantage is generally based on the tumor cytotoxicity of individual drugs or combinations of drugs [13]. Our earlier work showed a link between the use of cytotoxic chemotherapy with etoposide (VP-16) and the induction of an immune response against syngeneic murine leukemia in the intact host [16]. VP-16 is an immunosuppressive topoisomerase II-inhibiting drug which induces tumor cell apoptosis and is frequently used clinically to treat a variety of tumors [1, 3, 9, 10]. We have noted that the addition of cyclosporin A to VP-16 produces CD8 T lymphocyte-mediated tumor-specific immunity in mice bearing L1210 leukemia [17]. We have extended these experiments to a spontaneously arising non-carcinogen-induced neoplasm, Lewis lung cancer (3LL), and now report that surviving mice successfully treated with VP-16, in the absence of cyclosporin A, reject challenge with 3LL. In addition, results are presented to show that VP-16 modifies 3LL cells rendering them immunogenic. These findings are submitted to support the hypothesis that VP-16-induced cytotoxic changes include cellular membrane alterations in 3LL cells which are recognized by the immune system and cause rejection of this syngeneic lung tumor.

Superiority of cyclosporin A over PSC-833 in enhancement of VP-16 efficacy in murine tumors in vivo.

PSC-833, a non immunosuppressive analogue of cyclosporin A, is an effective modulator of the multidrug-resistant tumor phenotype. Since both PSC-833 and cyclosporin A also enhance the cytotoxicity of VP-16 against drug sensitive L1210 leukemia cells in vitro we compared these agents as modulators of VP-16 efficacy in vivo. Compared to VP-16 treatment alone both PSC-833 and cyclosporin A significantly altered the survival of L1210 leukemia-bearing BDF/1 mice and Lewis lung carcinoma-bearing C57/B1 mice. Cyclosporin A enhanced VP-16 efficacy whereas PSC-833 impaired VP-16 efficacy against these murine tumors. Possible reasons for these disparate effects are discussed.

Comparison of cyclosporin A, verapamil, PSC-833 and cremophor EL as enhancing agents of VP-16 in murine lymphoid leukemias.

Although verapamil, cyclosporin A. cremophor EL and PSC-833 are active as multidrug resistance modulators, there has been limited study of these compounds as possible chemotherapy enhancing agents against drug-sensitive tumors. We compared these agents as modifiers of VP-16 cytotoxicity in vitro and modifiers of VP-16 efficacy in vivo against drug-sensitive P388 and L1210 leukemias. Our study indicates that cyclosporin A enhances VP-16 cytotoxicity to a significantly greater extent than equimolar concentrations of verapamil or PSC-833. Although cremophor EL shows significantly greater activity than verapamil in VP-16 cytotoxicity enhancement in vitro, it is ineffective when added to VP-16 therapy of mice bearing L1210 leukemia.

Cyclosporin A/VP-16 produced immunity to L1210 leukemia: the participation of cytotoxic CD8 T-lymphocytes.

The role of the immune response in the chemotherapeutic cure of an intact host with neoplasia is not well defined. We have previously shown that the addition of cyclosporin A to VP-16 therapy of BDF/1 mice with L1210 leukemia produces immunity to leukemia in long-surviving host animals. We now characterize this immunity as being tumor specific and related to the participation of CD8 T-lymphocytes. Splenocytes derived from L1210 leukemia immune mice are cytotoxic to L1210 cells after in vitro restimulation, compared to splenocytes harvested from nonimmune control mice. This cytotoxicity is lost by CD8 T-lymphocyte depletion and persists in Ia antigen blocking experiments. Cytotoxicity is selective for L1210 leukemia compared to P388 leukemia, an alternate Ia antigen expressing methylcholanthrine-induced acute lymphoid leukemia, and L1210 leukemia-immune mice remain susceptible to P388 leukemia in vivo demonstrating specificity of the immune response generated by cyclosporin A/VP-16 therapy.

Enhancement by cyclosporin A of daunorubicin efficacy in Ehrlich ascites carcinoma and murine hepatoma 129.

Cyclosporin A abrogates pleiotropic drug resistance in certain experimental tumors. Its impact on drug-sensitive tumors has not been investigated. Our studies show that in drug-sensitive Ehrlich ascites carcinoma and hepatoma 129 cyclosporin A enhances daunorubicin inhibition of DNA synthesis in vitro and prolongs survival of host mice in vivo. Of particular interest is that cyclosporin A converts ineffective daunorubicin regimens into those which result in prolongation of host mice survival. Other agents known to reverse pleiotropic drug resistance are reported to exert their effects by increasing intracellular drug accumulation. In contrast, our studies of drug transport in drug-sensitive Ehrlich ascites carcinoma and hepatoma 129 show that cyclosporin A causes minimal enhancement of [3H]daunorubicin uptake without inhibition of [3H]daunorubicin efflux in both the presence and absence of interrupted active daunorubicin efflux. This suggests that the mechanism of action of daunorubicin enhancement by cyclosporin A in drug-sensitive tumors is not simply the result of increased intracellular daunorubicin accumulation. In vivo dosages of cyclosporin A in the current study are comparable to those which can be used with reasonable safety in humans. We conclude that cyclosporin A may be useful in the potentiation of anthracycline antibiotic therapy directed against drug-sensitive as well as drug-resistant tumors.

Albumin inhibition of the antileukemic activity of hydroxylated phenothiazines.

Trifluoperazine (TFP) shows cytotoxic activity against human acute lymphatic leukemia (ALL) in vitro. This activity is inhibited by increasing serum concentration and by albumin. Despite its in vitro activity, the drug is inactive in vivo. To determine if increased phenothiazine hydrophilicity could protect against albumin inhibition of antileukemic activity, we compared ALL cytotoxic median effective dose concentrations of a series of hydroxylated phenothiazines in 5% fetal bovine serum (FBS) and in 5% FBS supplemented with albumin. Albumin inhibits the activity of all drugs. A representative derivative 7,8-dihydroxychlorpromazine, although active in vitro, is inactive against L1210 and P388 murine leukemias in vivo.

Cyclosporin A corrects daunorubicin resistance in Ehrlich ascites carcinoma.

We have previously developed a daunorubicin resistant subline of Ehrlich ascites carcinoma (EA/DR) for studies on the reversal of daunorubicin resistance. The mean survival of untreated BALB/c mice bearing drug sensitive parental tumour (EA/DS) is 18.4 +/- 0.6 days, mice bearing EA/DS treated with five daily doses of 0.3 mg kg-1 daunorubicin greater than 60 days, and mice bearing EA/DR treated with the same daunorubicin regimen, 21.1 +/- 1.4 days. We now report complete reversal of daunorubicin resistance in EA/DR by cyclosporin A (CsA). The in vitro daunorubicin IC50, defined as that concentration of daunorubicin required to inhibit 50% of DNA synthesis, in EA/DR was 6.7 +/- 1.15 micrograms ml-1 compared to 2.8 +/- 0.72 micrograms ml-1 in EA/DS. This value was reduced to 2.8 +/- 0.52 and 2.1 +/- 0.10 micrograms ml-1 daunorubicin by 3.3 and 13.2 micrograms ml-1 CsA respectively, P less than 0.05. The MST of groups of host mice bearing EA/DR either untreated, treated with five daily doses of 0.3 mg kg-1 daunorubicin, treated with 80 mg kg-1 CsA in five divided daily doses or treated with combined daunorubicin-CsA were 19.0 +/- 1.0, 21.1 +/- 1.4, 24.0 +/- 2.6 and greater than 60 days respectively. The mean survival of groups of host mice bearing EA/DR treated with 5 mg kg-1 or 10 mg kg-1 CsA simultaneously with daunorubicin for five days was also greater than 60 days. These differences are highly significant.

Cyclosporin A reverses vincristine and daunorubicin resistance in acute lymphatic leukemia in vitro.

The development of drug resistance by tumor cells is a major obstacle to the cure of human malignancy. Cyclosporin A (CsA) completely reverses primary resistance to vincristine and cross resistance to daunorubicin in a pleiotropic drug-resistant subline of human T cell acute lymphatic leukemia. This subline is over 50-fold resistant to vincristine and fivefold resistant to daunorubicin. CsA has little effect on vincristine or daunorubicin activity in drug-sensitive parental leukemia and corrects daunorubicin resistance without altering cellular daunorubicin accumulation.

Verapamil potentiation of VP-16-213 in acute lymphatic leukemia and reversal of pleiotropic drug resistance.

Verapamil, the calcium-influx-blocking agent, has previously been shown to have favorable interactions with antineoplastic drugs. Our study of human T cell acute lymphatic leukemia (ALL) GM3639 indicates that verapamil enhances the in vitro cytotoxicity of VP-16-213 against drug-sensitive ALL by reducing the concentration of VP-16-213, resulting in 50% cell viability from 104.5 +/- 26.6 nM to 46.0 +/- 2.7 nM (P less than 0.05). The addition of verapamil to VP-16-213 treatment of BDF/1 mice bearing L1210 leukemia increases their mean survival from 21.2 +/- 3.6 to 50.4 +/- 4.3 days (P less than 0.01) and the survival of CD2F/l mice bearing P388 leukemia from 27.8 +/- 3.7 to 49.1 +/- 5.0 days (P less than 0.01). The 30-day survival is significantly increased in L1210 and P388 leukemia mice, and 60-day survival is significantly increased in P388 leukemic mice by verapamil. We developed a vincristine (VCR)-resistant subline of GM3639 T cell ALL, L23, by continuous exposure of drug-sensitive cells to VCR. This subline demonstrates pleiotropic cross resistance to VP-16-213 and daunorubicin. The addition of verapamil to VCR, to VP-16-213, and to daunorubicin completely restores responsiveness to these drugs, as indicated by the normalization of the VCR and VP-16-213 concentrations required for cytotoxicity and the concentration of daunorubicin required for inhibition of thymidine incorporation.