Synthesis of new coumarin tethered isoxazolines as potential anticancer agents.

A series of new coumarin tethered isoxazolines (7a-l) were synthesized and evaluated for their cytotoxic potency against human melanoma cancer cell line (UACC 903) as well as fibroblast normal cell line (FF2441). Preliminary results revealed that some of these coumarin tethered isoxazolines 7b, 7c, 7f and 7j exhibited significant antiproliferative effect against human melanoma cancer (UACC 903) with IC50 values of 8.8, 10.5, 9.2 and 4.5 µM respectively. However, compound 7c was non-toxic to normal human cells at the tested concentration. Further, we have chosen compound 7c to check its efficacy in Ehrlich Ascites Carcinoma animal model in-vivo for its antitumor and antiangiogenic properties. Our lead compound significantly reduced the cell viability, body weight, ascites volume and downregulated the formation of neovasculature such as regression of tumor volume. The present study indicates the scope of developing into potent anticancer drug in near future.

High Antimetastatic Activity of Platin Liposomal Form after Lyophilization and Storage.

Antimetastatic activity of Platin in lyophilized liposomes stored for 7 years after fabrication was evaluated. The main flaw of liposomes as vehicles for drug delivery to the tumors is their high affinity for the liver, which accumulates a great amount thereof. This property of liposomes can be used for adjuvant therapy of operable primary tumors metastasizing to the liver. It is shown on the model of mouse GA-1 tumor metastases in the liver that platinum(II) complex compound Platin in phosphatidylcholine-cholesterol liposomes, stored for 7 years after lyophilization, causes complete cure of 40% animals, while free Platin prolongs the lifespan of mice with tumors by only 31.7% vs. control (no treatment).

Could Caffeic Acid Phenethyl Ester Expand the Antitumor Effect of Tamoxifen in Breast Carcinoma?

Despite tamoxifen (TAM) is beneficial in treating a significant proportion of patients with breast cancer, many women still relapse after long-term therapy. Caffeic acid phenethyl ester (CAPE) is a component of honeybee propolis, with a plethora of important biological actions including anticancer activity. This study aimed to explore the cytotoxicity, the type of drugs interaction as well as the apoptotic and autophagic pathways of the combined treatment of TAM and CAPE in MCF-7 cells. Their antitumor activity and effect on survival of mice bearing Ehrlich tumor were also analyzed. The results showed synergistic cytotoxic effects, manifested by significant activation of apoptotic machinery, along with downregulation of protein levels of Bcl-2 and beclin-1, upon using the combination regimen. However, the ratio between microtubule-associated protein light chain 3-II and -I was not altered. Moreover, a decrease in vascular endothelial growth factor level was detected. Similarly, TAM + CAPE increased the life span of tumor-bearing animals and caused a marked regression in their tumor size and weight compared with those treated with either TAM or CAPE alone. In conclusion, CAPE relatively improved the anticancer activity of TAM in both in vitro and in vivo models via its apoptotic and angiostatic potentials.

Study of Antitumor Activity of Sodium Phenylbutyrate, Histon Deacetylase Inhibitor, on Ehrlich Carcinoma Model.

Antitumor activity of sodium phenylbutyrate was studied on 120 outbred female mice with transplanted Ehrlich ascites carcinoma. The animals received the drug in doses of 400, 800, and 1200 mg/kg with drinking water daily for 21 days. The antitumor effect was evaluated by tumor growth inhibition and lifespan prolongation. Phenylbutyrate in the dose of 800 mg/kg was most effective. The drug inhibited the tumor growth by 71%, prolonged the lifespan of animals by 28, and was low-toxic.

[Bacterial phagelysates and malignant tumor growth].

Anti-tumor preventive efficacy of E.coli phagelysate has been studied. Investigations were conducted on 2-3 months 48 male mice. Regimen of preventive vaccinations were: single - 0,25 ml phagelysate intraperitoneal injection, 3 days before Ehrlich carcinoma inoculation (1x10(6) tumor cells); 3 times vaccinations (0,25 ml, with 3 day intervals) 3, 6, and 9 days before inoculation of carcinoma; and 10 times (during 10 days, before inoculation of carcinoma). Treatment efficacy was evaluated according to the indices of cancer growth (development of cancer tissue, cancer growth inhibition percent, lifespan and survival percent). Experiments have shown that single and 3 times preventive vaccinations inhibited tumor development and delayed malignant growth, while, 10 times permanent vaccinations had no effects on cancer growth. Cancer growth inhibition percent in single and 3 times vaccinated animals were 58% on the average. Maximal lifespan in control group mice consisted 59 days. By the 125th day of cancer growth, at single vaccination 17% of mice were alive, while in 3 times vaccinated mice the survival percent was 25%. Anti-tumor potential of E.coli pagelysate supposedly could be explained by immunoregulatory properties of the preparation.

Sleep deprivation increases mortality in female mice bearing Ehrlich ascitic tumor.

OBJECTIVES: Sleep deprivation is a growing public health hazard, yet it is still under-recognized. Sleep disorders and disruption of sleep patterns may compromise the immune function and adversely affect host resistance to infectious diseases. This is a particular risk in cancer patients, who report a high frequency of sleep disturbances. The present study investigated the effects of sleep deprivation on the development of Ehrlich ascitic tumors (EAT) in female BALB/c mice. Our study also evaluated whether EAT would induce alterations in sleep pattern. Spleen lymphocyte cell populations and mortality were also quantified. METHODS: Female BALB/c mice were intraperitoneally inoculated with EAT cells. Immediately after the inoculation procedure, animals were sleep deprived for 72 h. Ten or 15 days after inoculation, the number of tumoral cells was quantified and the lymphocytic cell population in the spleen was characterized by flow cytometry. In addition, the effect of sleep deprivation on EAT-induced mortality was quantified and the influence of EAT on sleep patterns was determined. RESULTS: Sleep deprivation did not potentiate EAT growth, but it significantly increased mortality. Additionally, both EAT and sleep deprivation decreased frequencies of splenic CD4+, CD8+ and CD19+ cells. With respect to sleep patterns, EAT significantly enhanced paradoxical sleep time. CONCLUSIONS: Although sleep deprivation did not potentiate EAT growth, it decreased the survival of female tumor-bearing mice.

Antitumor activity of cucumarioside A2-2.

BACKGROUND: The cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. However, the molecular mechanism(s) of the antitumor action of triterpene glycosides on cancer cells remain unclear. This article reports a continuation of investigations of triterpene glycoside cucumarioside A2-2 isolated from the Far-Eastern sea cucumber Cucumaria japonica. It describes a study of glycoside anticancer activity in vivo and glycoside interaction with mouse Ehrlich carcinoma cells in vitro. METHODS: The cytotoxicity of cucumarioside A2-2 and its effect on apoptosis, the cell cycle, DNA biosynthesis and p53 activity, and glycoside anticancer action against Ehrlich carcinoma cells were studied. RESULTS: Cucumarioside A2-2 influences tumor cell viability at micromolar concentrations. The EC50 for glycoside estimated by nonspecific esterase assay and MTT assay was 2.1 and 2.7 µM, respectively. Cucumarioside A2-2 at a subcytotoxic range of concentrations exhibits a cytostatic effect by blocking cell proliferation and DNA biosynthesis in the S phase. It may induce apoptosis in tumor cells in a caspase-dependent way, bypassing the activation of the p53-dependent segment. CONCLUSION: The anticancer and proapoptotic properties of cucumarioside A2-2 may be due to direct interaction of the glycoside with tumor cells. The in vivo anticancer effect of cucumarioside A2-2 may be associated with the ability of the drug to arrest the cell cycle in the synthetic phase and induce programmed tumor cell death.

Dynamics of antitumor resistance after cyclophosphamide injection.

Antitumor resistance decreased in mice 24 h after injection of cyclophosphamide in a dose of 100 mg/kg. This was seen from more rapid growth of Ehrlich's ascitic carcinoma transplanted intraperitoneally 24 h after cyclophosphamide injection and 17% reduction of the lifespan of mice with tumors. Three, 7, 10, 14, and 22 days after cyclophosphamide injection, the antitumor resistance increased and the lifespan of animals with Ehrlich's ascitic carcinoma transplanted at the corresponding periods increased by 20, 14, 42, 29, and 36%, respectively, in comparison with mice with transplanted tumor not injected with the drug. Injection of cyclophosphamide 1 day after tumor transplantation prolonged of the lifespan of animals with tumors by 76%.

Paullinia cupana Mart. var. sorbilis, guarana, increases survival of Ehrlich ascites carcinoma (EAC) bearing mice by decreasing cyclin-D1 expression and inducing a G0/G1 cell cycle arrest in EAC cells.

The objective of this work is to report the antiproliferative effect of P. cupana treatment in Ehrlich Ascites Carcinoma (EAC)-bearing animals. Female mice were treated with three doses of powdered P. cupana (100, 1000 and 2000 mg/kg) for 7 days, injected with 10(5) EAC cells and treated up to day 21. In addition, a survival experiment was carried out with the same protocol. P. cupana decreased the ascites volume (p = 0.0120), cell number (p = 0.0004) and hemorrhage (p = 0.0054). This occurred through a G1-phase arrest (p < 0.01) induced by a decreased gene expression of Cyclin D1 in EAC cells. Furthermore, P. cupana significantly increased the survival of EAC-bearing animals (p = 0.0012). In conclusion, the P. cupana growth control effect in this model was correlated with a decreased expression of cyclin D1 and a G1 phase arrest. These results reinforce the cancer therapeutic potential of this Brazilian plant.

Antitumor efficacy and amelioration of oxidative stress by Trichosanthes dioica root against Ehrlich ascites carcinoma in mice.

CONTEXT: Trichosanthes dioica Roxb. (Cucurbitaceae) is a dioecious climber, traditionally used in India for several medicinal purposes. OBJECTIVE: The present study assessed the hydroalcoholic extract of T. dioica root (TDA) for antitumor effect and antioxidant influence against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. METHODS: Twenty four hours after intraperitoneal inoculation of tumor (EAC) cells in mice, TDA was administered at 5 and 10 mg/kg body weight daily for 9 consecutive days. On the 10th day, half of the mice were sacrificed for estimation of tumor proliferation, hematological, and liver antioxidant parameters viz. lipid peroxidation, reduced glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT); and the rest were kept alive for assessment of increase in life span. The antitumor effect of TDA was assessed by evaluating tumor weight, tumor volume, packed cell volume, viable and non-viable tumor cell counts, median survival time and percentage increase in life span of EAC bearing mice. RESULTS AND DISCUSSION: TDA exhibited dose dependent and significant (p < 0.001) decrease in tumor weight, tumor volume, packed cell volume and viable cell count and extended the life span of EAC bearing hosts. Hematological profiles were significantly (p < 0.001) restored near to normal in TDA treated mice as compared to EAC control. TDA treatment significantly (p < 0.001) modulated the aforesaid liver antioxidant parameters as compared to EAC control. CONCLUSION: The present study demonstrated that TDA possessed promising antitumor efficacy in mice, plausibly mediated by amelioration of oxidative stress by multiple mechanisms.

Enhanced anticancer activity of combined treatment of imatinib and dipyridamole in solid Ehrlich carcinoma-bearing mice.

The current study was designed to evaluate potential enhancement of the anticancer activity of imatinib mesylate (IM) with dipyridamole (DIP) and to investigate the underlying mechanisms of the combined therapy (IM/DIP) to reduce hepatotoxicity of IM in solid Ehrlich carcinoma (SEC)-bearing mice. SEC was induced in female albino mice as a model for experimentally induced breast cancer. Mice were randomly divided into seven groups (n = 10): SEC vehicle, IM50 (50 mg/kg), IM100 (100 mg/kg), DIP (35 mg/kg), a combination of IM50/DIP and IM100/DIP. On day 28th, mice were sacrificed and blood samples were collected for hematological studies. Biochemical determination of liver markers was evaluated. Glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT) and alkaline phosphatase (ALP) levels were assessed. In addition, MDR-1 gene expression and immunohistochemical staining of BAX and BCL-2 was done. Also, in vitro experiment for determination of IC50 of different treatments and combination index (CI) were assessed in both MCF-7 and HCT-116 cell lines. IM- and/or DIP-treated groups showed a significant reduction in tumor volume, weight, and serum levels of SGOT, SGPT, and AIP compared to vehicle group. In addition, reduction of VEGF, Ki67, and adenosine contents was also reported by treated groups. Also, IM/DIP combination showed lower IC50 than monotherapy. Combination index is less than 1 for IM/DIP combination in both cell lines. DIP as an adjuvant therapy potentiated the cytotoxic effect of IM, ameliorated its hepatic toxicity, and showed synergistic effect with IM in vitro cell lines. Furthermore, the resistance against IM therapy may be overcome by the use of DIP independent on mdr-1 gene expression.

Synthesis, antitumor evaluation, molecular modeling and quantitative structure-activity relationship (QSAR) of some novel arylazopyrazolodiazine and triazine analogs.

The synthesis, in vivo and in vitro antitumor evaluation, and QSAR studies of some novel pyrazole analogs against Ehrlich Ascites Carcinoma (EAC) cells were described. In vitro results revealed that compounds 10, 6 and 4 were the most potent analogs against EAC, respectively. Moreover, in vivo evaluation of compounds 6 and 10 proved their capability to normalize the blood picture in comparison to 5-FU, a well known anticancer drug. These novel pyrazole analogs were molecularly designed with the goal of having significant potent cytotoxic effect against EAC cells. To develop a QSAR model capable of identifying the key molecular descriptors associated with the biological activity of the novel pyrazole analogs and predicting the cytotoxic effect for other novel pyrazole analogs against EAC cells, different QSAR models, using different physicochemical and topological molecular descriptors, were developed. Different molecular descriptors were predicted solely from the chemical structures of 16 pyrazolo-diazine and triazine analogs following the prediction of the equilibrium molecular geometry of each analog at the DFT level using B88-LYP functional energy and double zeta valence polarized (DZVP) basis set. It was found that dipole moment, excitation energy, the energy value of LUMO, solvent accessible surface area, and heat of formation were the key molecular descriptors in descriping the cytotoxic effect of those compounds against EAC.

Solvatochromism, DNA binding, antitumor activity and molecular modeling study of mixed-ligand copper(II) complexes containing the bulky ligand: bis[N-(p-tolyl)imino]acenaphthene.

Four mixed-ligand copper(II) complexes of the nitrogen ligand bis[N-(p-tolyl)imino]acenaphthene 1 (p-Tol-BIAN). These complexes, namely [Cu(p-Tol-BIAN)2](ClO4)2 2, [Cu(p-Tol-BIAN)(acac)](ClO4) 3, [Cu(p-Tol-BIAN)Cl2] 4 and [Cu(p-Tol-BIAN)(AcOH)(2)](ClO4)2 5, were prepared and characterized. Solvatochromism of the novel copper complexes in various solvents has been studied. Molecular mechanics (MM+) and molecular dynamic simulations have been performed to learn more about the solvatochromic behaviour and the DNA binding affinity of these complexes.

Anticancer potential of Amaryllidaceae alkaloids evaluated by screening with a panel of human cells, real-time cellular analysis and Ehrlich tumor-bearing mice.

In this study, twenty-two Amaryllidaceae alkaloids were screened for their anticancer potential. All isolates were evaluated for antiproliferative activities on a panel of 17 human cell types of different tissue origin using WST-1 assay. In addition, we determined the antiproliferative effect with a real-time cell analysis xCELLigence system. Thereafter, to evaluate the barely known in vivo anticancer potential of the most potent molecule haemanthamine, a preliminary study was performed using an Ehrlich tumor-bearing mice model. The results showed that haemanthamine, lycorine and haemanthidine exerted the highest antiproliferative activity. The mean growth percent (GP) value after a single-dose 10 µM treatment was for haemanthamine 21%, for lycorine 21% and for haemanthidine 27% that of untreated control cells (100%). Furthermore, haemanthamine, lycorine and haemanthidine exhibited significant cytotoxicities against all the tested cell lines with individual IC50 values in the micromolar range. Dynamic real-time measures of impedance by xCELLigence indicated that these three compounds suppress cell proliferation after 10 h of treatment at a concentration of 10 µM or higher. Regrettably, in a follow-up in vivo antitumor activity study, haemanthamine showed no statistically significant reduction in the tumor size with no prolongation of survival time of Ehrlich tumor-bearing mice. Taken together, these results provide a new clue and guidance for exploiting Amaryllidaceae alkaloids as anticancer agents.

Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment.

The leaves of Chenopodium ambrosioides L. [Chenopodiaceae] ('mastruz') have been indicated for the treatment of several diseases, among which the cancer. There are no results focusing the effect of C. ambrosioides treatment on tumor development in vivo. The aim of this study was to investigate the effect of treatment with C. ambrosioides on Ehrlich tumor development. Swiss mice were treated by intraperitoneal route (i.p.) with hydroalcoholic extract from leaves of C. ambrosioides (5 mg/kg) or with PBS (control group) 48 h before or 48 h later the Ehrlich tumor implantation. The tumor cells were implanted on the left footpad (solid tumor) or in the peritoneal cavity (ascitic tumor). To determine the solid tumor growth, footpad was measured each 2 days until the fourteenth day, when the feet were weighed. Ascitic tumor development was evaluated after 8 days of tumor implantation by quantification of the ascitic fluid volume and tumor cell number. The i.p. administration of C. ambrosioides extract before or after the tumor implantation significantly inhibited the solid and ascitic Ehrlich tumor forms. This inhibition was observed in ascitic tumor cell number, in the ascitic volume, in the tumor-bearing foot size and foot weight when compared to control mice. The treatments also increased the survival of tumor-bearing mice. In conclusion, C. ambrosioides has a potent anti-tumoral effect which was evident with a small dose and even when the treatment was given two days after the tumor implantation. This effect is probably related with anti-oxidant properties of C. ambrosioides.

Trans fatty acids in tumor development and the host survival.

Because trans fatty acids exist in the American diet and their relationship to cancer incidence has been proposed, studies were designed to investigate their possible role in the development of Ehrlich tumor cells in inbred CBA mice. Feeding elaidic acid (18:1 delta 9, trans) to Ehrlich ascites tumor-bearing animals resulted in tumors that incorporated more thymidine into their cells and into their acid-insoluble fraction as compared with those grown in animals fed the natural oleic acid (18:1 delta 3, cis)-rich diet. Elaidic acid diet feeding at 5% in the diet also resulted in a reduction in the host survival rate. This reduction ranged from 23 to 45%.

Effect of altered lighting regimens, time-limited feeding, and presence of Ehrlich ascites carcinoma on the circadian rhythm in DNA synthesis of mouse spleen.

The objectives of the series of experiments described were (a) to determine whether there was a circadian rhythm in the incorporation of tritiated thymidine into DNA of the spleen in mice kept on a conventional light-dark cycle and fed ad libitum, (b) to study the effect that different light-dark cycles and a time-limited feeding schedule had on this circadian rhythm, (c) to ascertain what effect the presence of an 8-day Ehrlich ascites carcinoma (EAC) had on the rhythm in DNA synthesis in the spleen and what effect the EAC had on the circadian rhythm in the mitotic index in the corneal epithelium, and (d) to determine whether there was a circadian rhythm in the duration of life-span in mice bearing the EAC. A circadian rhythm in the incorporation of tritiated thymidine into DNA of the mouse spleen consistently was characterized by a peak during the nocturnal phase and a trough during the diurnal phase of the 12-hr light-12-hr dark cycle. In animals bearing an 8-day EAC, the rhythm in DNA synthesis in the spleen was phase-shifted, its wave form was changed, and the overall 24-hr mean was increased significantly. The phasing of the rhythm in EAC-bearing mice was not reproducible. This finding demonstrated that the presence of the EAC severely altered the natural rhythm in DNA synthesis in the spleen and resulted in a rhythmic pattern which was constantly changing. The presence of an 8-day EAC, however, had no effect on the amplitude, overall level, or the phasing of the circadian rhythm in the mitotic index in the cornea. A staggered light-dark cycle of 2 weeks duration did not completely phase-shift the DNA synthesis rhythm in the spleen but did completely phase-shift the rhythm in the mitotic index in the corneal epithelium. In mice subjected to a daily feeding period limited to 4 hr, the rhythm in DNA synthesis in the spleen, in both EAC-bearing and non-EAC bearing mice, was phase-shifted such that the peak occurred during the time of feeding and the trough occurred prior to the feeding period. The rhythm in the mitotic index in the cornea was not phase-shifted or altered in any way by the feeding schedule and thus remained fixed to the light-dark cycle. The DNA synthesis rhythm in the normal spleen demonstrated a phasing very similar to the circadian rhythm in the length of survival in mice challenged with EAC and the circadian rhythm in DNA synthesis in the normal thymus.

Macrophages Reprogrammed In Vitro Towards the M1 Phenotype and Activated with LPS Extend Lifespan of Mice with Ehrlich Ascites Carcinoma.

BACKGROUND The majority of tumors trigger macrophage reprogramming from an anti-tumor M1 phenotype towards a pro-tumor M2 phenotype. The M2 phenotype promotes tumor growth. We hypothesized that increasing the number of M1 macrophages in a tumor would limit carcinogenesis and extend the lifespan of the tumor host. The aim of this study was to verify this hypothesis in Ehrlich ascites carcinoma (EAC). The objectives were to evaluate effects of 1) EAC on a macrophage phenotype and NO-producing macrophage activity in vivo; 2) ascitic fluid from mice with EAC on a macrophage phenotype and NO-producing macrophage activity in vitro; and 3) in vitro reprogrammed M1 macrophages on lifespan of mice with EAC. MATERIAL AND METHODS The study was conducted using C57BL/6J mice. RESULTS Concentration of nitrite, a stable NO metabolite and an index of NO production, was measured spectrophotometrically. Shifts of macrophage phenotype were assessed by changes in NO production as well as by amounts of CD80, a marker of M1 phenotype, and CD206, a marker of M2 phenotype. The CD markers were measured by flow cytometry. Macrophages were reprogrammed towards the M1 phenotype using two reprogramming factors: 0% FBS and 20 ng/ml IFN-γ. The study results showed that 1) EAC inhibited the macrophage NO production in vivo and reprogrammed macrophages towards the M2 phenotype; 2) ascitic fluid of mice with EAC inhibited the macrophage NO production in vitro and reprogrammed macrophages towards the M2 phenotype; and 3) injection of in vitro reprogrammed M1 macrophages into mice with EAC significantly increased the lifespan of mice. CONCLUSIONS These findings suggest that promising biotechnologies for restriction of tumor growth could be developed based on the in vitro macrophage reprogramming.

[FUNCTIONAL ACTIVITY OF EHRLICH CARCINOMA CELLS AFTER TREATMENT WITH HYBRID NANOCOMPLEXES CONTAINING ORTHOVANADATES OF RARE-EARTH ELEMENTS, CHOLESTEROL AND LUMINESCENT DYE].

Tumor development is the consequence of expanding the population of low differentiated cells with unlimited self-maintenance potential, i.e. cancer stem cells (CSCs). Application of new forms of nanocomposites capable of binding to CSCs and inducing the tumor destruction is perspective direction for treating this pathology. There have been developed the methods of obtaining hybrid nanocomplexes containing rare-earth orthovanadates GdYVO4:Eu³âº, cholesterol and luminescent dye Dil. By immune fluorescence method using monoclonal antibodies to CD44, CD24, CD117 and Sca-1 markers there has been established the change in the ratio of tumor progenitors of various differentiation levels in a general pool of Ehrlich carcinoma (EC) after treatment with hybrid nanocomplexes. Essential reduction in the concentration of the most tumorogenic CD44high cells with simultaneous rise in the number of CD117⁺-cells resulted in an increased index of CD44high/CD117⁺ ratio. It has been demonstrated that application of hybrid nanocomplexes suppressed the tumor growth almost by 80%. The value of cooperative interactions of the cells with different phenotype signs in tumor sites has been proved. The index of CD44high/CD117⁺ ratio can be used as one of diagnostic and prognostic parameters of development and inactivation rate of tumor process when using different types of anti-tumor therapy.

Effect of combined ascorbic acid and B-12 on survival of mice with implanted Ehrlich carcinoma and L1210 leukemia.

A combination of dehydroascorbic acid and hydroxycobalamin (vitamin B-12) inhibited mitoses of tumors in mice. The present study was performed to test the effect of these vitamins on the survival of mice bearing carcinomas and leukemias. In each assay 40 mice received 0.1 mL ip tumor cells (x10(5)). After 24 h, 20 mice were injected with 0.2 mL (0.4 g/kg body wt) of the vitamins daily for 10 d. All controls died by day 19, but greater than 50% of the treated mice were alive after 60 d. In vitro findings revealed inhibition of mitoses in L1210 leukemia cells, but not in normal L929 cells. In recent research with cobalt-ascorbate plus vitamin C, we demonstrated that when B-12 is combined with vitamin C, the cobalt nucleus of B-12 attaches to a carbon on vitamin C, forming cobalt ascorbate. Tests proved that cobalt ascorbate plus vitamin C also inhibited tumor cells.