ascorbic-acid and Leukemia-P388

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.

Topics: Animals; Antineoplastic Agents; Antioxidants; Ascorbic Acid; ATP Binding Cassette Transporter, Subfamily B, Member 1; Catalase; Docosahexaenoic Acids; Doxorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; gamma-Linolenic Acid; Glutathione Peroxidase; Glutathione Transferase; Leukemia P388; Mice; Proline; Superoxide Dismutase; Thiocarbamates; Tumor Cells, Cultured

In recent years there has been a growing interest in the therapeutic application of L-ascorbic acid (AA) and its derivatives as anticancer agents. AA is a gamma-crotonolactone derivative with reactive hydroxyl groups at the 2- and 3-positions and an ethylene glycol substitution at the 4-position. Despite the various reports on AA toxicity, no work has been reported underlying the critical chemical structural features for its activity. The present study addresses this question. We tested in vivo, using malignant leukemia cell line P388D1, (i) L-AA and its isomers, (ii) substitution at the 2-position: -PO4, -SO4, O-Me, O-octadecyl, (iii) substitution at the 6-position: -PO4, -SO4, -palmitate, -stearate, (iv) substitution at the 2,6-position: dipalmitate, (v) 6-deoxy derivative: -Cl, -Br, -NH2 and (vi) dihydroxy gamma-crotonolactone with substitutions at the 4-position: -H, -CH3, -CH2-CH3 and -CH=CH2. L-AA and its isomers were very cytotoxic even at very low concentration. All 6-substituted and 6-deoxy derivatives were as toxic as AA. However, 2-substituted and 2,6-disubstituted AA derivatives were non-toxic. Interestingly, dihydroxy gamma-crotonolactone with or without substitution at the 5-position also exhibited toxicity. These results suggest that the underlying criterion for AA toxicity resides in dihydroxy gamma-crotonolactone moiety. Either substitution in the hydroxy groups or saturating the double bond render the molecule inactive.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Leukemia P388; Mice; Structure-Activity Relationship; Tumor Cells, Cultured

In recent years L-ascorbic acid (AA) and its isomers have raised considerable interest as anticancer agents, although the mechanism has remained largely unknown. AA isomers are nearly identical in their physical and chemical properties but differ widely in their biological properties. AA, a lactone sugar, has a number of reactive positions, especially at 2- and 6-. Although there are a number of reports on the cytotoxic effect of AA and its isomers on malignant and nonmalignant cell lines, no work has been reported on the comparative effects of substitutions at these active sites. This study, then, investigates the comparative cytotoxicity of such substitutes on the malignant leukemia P388 cell line in culture. We tested a series of 2-, 6- and 2,6- disubstituted AA-derivatives, comprising the following: i) substitution at 2-position: -PO4, -SO4, O-Me, O-octadecyl; ii) substitution at 6-position: -PO4, -SO4, -palmitate, -stearate; and iii) substitution at 2,6-position: -dipalmitate. About 50,000 P388 cells/ml were incubated with and without AA derivatives in a final concentration of 1000, 500, 100, 10 and 1 microg/ml in triplicate and counted after 72 hrs. All 2-substituted and the 2,6-substituted AA derivatives tested were nontoxic and ineffective in preventing cell growth. In contrast, all 6-substituted AA derivatives were very toxic at all levels, even at the lowest concentration. These results suggest that substitution at 2-, 6- and 2,6-positions in AA have a different effect on toxicity. The 2-, and 2,6-substituted AA derivatives are stable compounds, resistant to hydrolysis which render them inactive. The cytotoxicity of the 6-substituted derivatives may be explained by one of the following mechanisms, yet to be explored: i) the hydrolysis rate may differ; or ii) the chemical structure itself may affect toxicity. Further studies are in progress to understand the mechanism.

Topics: Animals; Ascorbic Acid; Cell Survival; Isomerism; Leukemia P388; Mice; Structure-Activity Relationship; Tumor Cells, Cultured

The antitumour, antimetastatic and antileukemic effect of cyclophosphane and adriamycin in combination with vitamins A. E. C was studied according to the scheme developed by the authors. The preliminary administration of vitamins was established to intensify the effect of cytostatics and to lower considerably their toxic action. Cyclophosphane proved to be more effective relative to the Lewis lung carcinoma than adriamycin.

Topics: Animals; Ascorbic Acid; Cyclophosphamide; Doxorubicin; Drug Therapy, Combination; Leukemia L1210; Leukemia P388; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasms, Experimental; Sarcoma, Experimental; Vitamin A; Vitamin E

The toxicity of daily subcutaneously applied 500 mg/kg N-methylformamide (NMF) during a period of 8 days in female CD-mice was ameliorated when 100 mg/kg sodium ascorbate, 60 mg/kg menadione bisulfite or 80 mg/kg pyridoxal hydrochloride were applied simultaneously. The comparison of the daily s.c. application of 360 mg/kg NMF with the intermittent s.c. injection of 720 mg/kg NMF with an interval of 48 h in P 388 leukemia showed that the daily application of NMF induced an increase of life span of 82% whereas the intermittent schedule effected a 142% increase of life span. The simultaneous combination of 360 mg/kg NMF with 60 mg/kg sodium ascorbate applied daily caused a 133% increase of life span and the simultaneous combination of 360 mg/kg NMF with 30 mg/kg menadione sodium bisulfite lead to a 126% increase of life span. The combined daily s.c. application of 360 mg/kg NMF with 30 mg/kg pyridoxal hydrochloride induced only a minimal difference compared to the daily application of 360 mg/kg NMF alone. The combination of 720 mg/kg NMF with 120 mg/kg sodium ascorbate applied in intervals of 48 h showed a 164% increase of life span. In advanced M 5076 sarcoma the daily s.c. application of 360 mg/kg NMF effected a 82% increase of life span and the combination of 360 mg/kg NMF with 60 mg/kg sodium ascorbate effected a 135% increase of life span.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Drug Synergism; Female; Formamides; Leukemia P388; Life Expectancy; Mice; Pyridoxal; Sarcoma, Experimental; Vitamin K; Vitamin K 3

The present study was conducted to clarify the mechanism responsible for enhancement of the anti-melanoma activity of levodopa methylester by supplemental ascorbate in vivo. 5-Hydroxydopa, a known cytotoxic agent and the major metabolite formed from levodopa in the presence of ascorbate and mushroom tyrosinase in vitro, was assessed for its antitumor activity against i.p. and s.c. inoculated B16 melanoma, P388 leukemia, and L1210 leukemia in mice with and without supplemental ascorbate. Treatment with 5-hydroxydopa failed to significantly increase survival of mice bearing i.p. or s.c. pigmented and non-pigmented B16 melanomas even though it inhibited local tumor growth. Treatment increased survival of both P388 and L1210 leukemias, and this increase was more pronounced in mice bearing i.p. tumors than in mice bearing s.c. tumors. This treatment significantly decreased final tumor weight of both leukemias implanted s.c., and inhibited ascites formation in mice inoculated with i.p. tumors. Ascorbate supplementation decreased or abrogated the effect of 5-hydroxydopa on survival in mice bearing i.p. or s.c. leukemia tumors and decreased survival relative to control mice bearing i.p. or s.c. pigmented and s.c. non-pigmented tumors. It did not affect survival of treated mice bearing i.p. non-pigmented melanoma tumors. Ascorbate supplementation did not modify the effect of 5-hydroxydopa treatment on primary s.c. tumor growth in mice bearing melanoma or leukemia tumors nor did it affect ascites formation in treated mice bearing i.p. leukemia tumors. The lack of correlation between the observed inhibition of primary tumor growth and the absence of an effect on survival in 5-hydroxydopa treated mice bearing i.p. melanoma may relate to an inability of this drug to interfere with tumor metastasis. These data argue against a role for 5-hydroxydopa as a metabolically derived cytotoxic formed in situ during concurrent treatment with levodopa methylester and supplemental ascorbate.

Topics: Animals; Ascorbic Acid; Dihydroxyphenylalanine; Drug Interactions; Female; Leukemia L1210; Leukemia P388; Male; Melanoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA

The transplantable murine ascites tumors, P388 leukemia and Ehrlich carcinoma, demonstrated complete inhibition of mitotic activity after treatment with dehydroascorbic acid in mice. Citric acid at the same pH value (2.4), however, showed no diminution of mitoses. Microscopic examination of the stained ascites exudate taken from the mice treated with 10 mg dehydroascorbic acid revealed few tumor cells and a pronounced increase in white blood cells, whereas the ascites tumor exposed to citric acid appeared normal.

Topics: Animals; Ascorbic Acid; Carcinoma, Ehrlich Tumor; Cell Division; Citrates; Citric Acid; Dehydroascorbic Acid; Leukemia P388; Leukemia, Experimental; Leukocyte Count; Mice; Mitosis

Topics: Animals; Ascorbic Acid; Carcinoma, Ehrlich Tumor; Drug Therapy, Combination; Leukemia P388; Leukemia, Experimental; Mice; Mice, Inbred Strains; Vitamin B 12