Biomedical subjects

B K Sinha

Publications and source records attributed to B K Sinha.

At least 55 records · Page 3Linked to original sources

PubMed1991

Doxorubicin-induced cross-resistance to tumor necrosis factor (TNF) related to differential TNF processing.

To evaluate whether cells selected for doxorubicin resistance were cross-resistant to tumor necrosis factor, the effects of doxorubicin and recombinant human tumor necrosis factor-alpha (TNF) on doxorubicin-sensitive (WT) and 40-fold doxorubicin-resistant (40F) MCF-7 cell proliferation were assessed. The median dose (MD) for doxorubicin was 14.5 nM for WT cells and 474 nM for 40F cells. The MD for TNF was 0.18 nM for WT cells, while 40F cells were highly resistant to TNF concentrations up to 60 nM. Doxorubicin and TNF in combination were synergistic against WT cells, but not 40F cells. Glutathione depletion by buthionine sulfoxamine sensitized WT cells threefold to TNF, with no change in their response to doxorubicin, while 40F cells showed a twofold increase in doxorubicin sensitivity, with no apparent change in their resistance to TNF. No significant differences in TNF receptor number, Kd, or capacity for TNF internalization were noted between the two cell types. WT cells produced a single 15 kDa TNF degradation product, while the 40F cells produced three lower molecular weight degradation products. We conclude that cross-resistance to TNF in doxorubicin-resistant MCF-7 cells may be explained in part by altered TNF degradation.

Buthionine Sulfoximine↗

PubMed1991

Cytochrome P-450- and peroxidase-dependent activation of procarbazine and iproniazid in mammalian cells.

Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metabolized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.

Biotransformation↗

PubMed1991

Selenium-dependent glutathione peroxidase expression is inversely related to estrogen receptor content of human breast cancer cells.

The absence of estrogen receptors (ER) in human breast tumors has been associated with a poorer prognosis compared to patients with ER positive breast cancer. Previous studies from our laboratory have shown that a multidrug resistant human breast cancer cell line selected for resistance to Adriamycin (ADR) exhibited markedly increased expression of both the pi class glutathione S-transferase (GST-pi) and the selenium-dependent glutathione peroxidase. These studies also revealed that the ER status was inversely related to the expression of GST-pi in six human breast cancer cell lines and primary tumor specimens. In the present study, we have examined the relationship between ER status and several biological properties of these cells, including their levels of glutathione peroxidase (GSH-Px) and catalase expression, their capacity to generate toxic hydroxyl radicals (degrees OH) by redox cycling of ADR, and their sensitivities to the cytotoxic effects of ADR and the oxidant, H2O2. Our results show that expression of GSH-Px, but not catalase, is inversely related to the ER status in these cell lines. Formation of the degree OH induced by treatment of cells with ADR was inversely proportional to the GSH-Px activity in these cell lines, and thus directly related to the ER status. Sensitivity of these cells to ADR or to H2O2, however, was not consistently related to ER status, GSH-Px, or catalase activity, or to ADR induced degree OH radical formation. These results indicate that these parameters are not predictive of cellular susceptibility to oxidative damage in these cell lines under the conditions studied.

Blotting, Northern↗

PubMed1990

Copper ion-dependent oxy-radical mediated DNA damage from dihydroxy derivative of etoposide.

The dihydroxy etoposide, a metabolite of the clinically active anticancer drug, VP-16, induced extensive DNA damage in the presence of copper ions. While superoxide dismutase was without any effect on the DNA damage, catalase and inhibitors of free hydroxyl radicals inhibited the DNA degradation, indicating that hydroxyl radicals were responsible for this drug-Cu-dependent DNA damage.

Animals↗

PubMed1990

Synergistic activity of suramin with tumor necrosis factor alpha and doxorubicin on human prostate cancer cell lines.

We evaluated the action of suramin, doxorubicin, and tumor necrosis factor alpha (TNF-alpha) on the testosterone-responsive human prostate cell line LNCaP and on the testosterone-independent human prostate cell line PC-3. The synergistic action of these agents in combination was tested by the Chou and Talalay method (quantitative analysis of dose-effect relationships) to determine whether in vitro doses were active at levels safely achieved in vivo. The action of suramin was potentiated threefold by doxorubicin for the PC-3 line and seven-fold by doxorubicin for the LNCaP line. Both the suramin-TNF-alpha and the doxorubicin-TNF-alpha combinations showed synergistic action against the LNCaP line. Synergistic activity was noted at drug concentrations routinely achieved clinically. This study demonstrates that suramin, doxorubicin, and TNF-alpha are active agents against prostate cancer cell lines and that their activity can be enhanced when they are used in combination.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1990

DNA interstrand cross-link and free radical formation in a human multidrug-resistant cell line from mitomycin C and its analogues.

A subline of the human breast tumor cell line (MCF-7), selected for resistance to Adriamycin and having the multidrug resistance phenotype, also developed significant cross-resistance to mitomycin C and its two analogues, BMY 25282 and BMY 25067. Because mitomycin C and the analogues contain both quinone and aziridine moieties, the mechanism of tumor cell kill is thought to involve alkylation and cross-linking of DNA molecules, hence they are not expected to show cross-resistance to cells selected for resistance to a DNA intercalator. Studies to understand this novel observation show that the resistant MCF-7 cells form significantly less hydroxyl radical and DNA cross-linking in the presence of mitomycin C and BMY 25282 than the sensitive cells. Although BMY 25067 formed less free radicals in the resistant cells, similar to the other two drugs, the formation of DNA cross-links was identical in both cell lines, indicating a somewhat different mechanism of tumor cell kill by this analogue. DNA cross-link formation increased slightly with time in the sensitive cells while there was a small decrease in the resistant cells. This difference in the formation of toxic intermediates appeared to result from enhanced detoxification of reactive species (hydrogen peroxide and alkylating intermediates) as a result of significantly higher glutathione peroxidase (14-fold) and glutathione S-transferase (44-fold) activities in the resistant cell line. These events, i.e., free radical formation and DNA alkylation, showed a good correlation with the cytotoxicity in drug-sensitive cells, indicating that both mechanisms contribute to cell killing of human breast tumor cells.

Alkylating Agents↗

PubMed1990

Role of oxygen free radical formation in the mechanism of menogaril resistance in multidrug resistant tumor cells.

The mechanisms of action and resistance to menogaril, a clinically active anthracycline antitumor drug, were evaluated in sensitive and doxorubicin-selected multidrug resistant human breast tumor (MCF-7) cell lines. While MCF-7/ADRR cells were highly resistant (250-500-fold) to doxorubicin, they displayed only marginal resistance (10-fold) to menogaril. In contrast to doxorubicin, the mechanism of resistance to menogaril in these cells does not involve differential inhibition of DNA synthesis as measured by thymidine incorporation. P-170-glycoprotein-dependent drug transport did not contribute to resistance as there was no difference in the accumulation and retention of menogaril by sensitive and resistant cell lines. However, there was a 2-fold decrease in oxygen free radical formation in the resistant cells, compared to sensitive cells, in the presence of menogaril. Since resistant cells contain 12-fold higher glutathione peroxidase activity than the parental sensitive cells, the detoxification of hydrogen peroxide may be responsible for the decreased free radical formation and thus, may play a role in the resistance to menogaril.

Antineoplastic Agents↗

PubMed1990

Structure-activity relations, cytotoxicity and topoisomerase II dependent cleavage induced by pendulum ring analogues of etoposide.

The cytotoxicity of etoposide and its analogues, dihydroxy (DHVP), o-quinone (VP-Q) and o-methyl (VP-OMe), was evaluated in human breast (MCF-7) and HL60 tumour cells. Although less potent than etoposide, both DHVP and VP-Q were cytotoxic to these cells. However, VP-OMe was inactive. Studies with purified topoisomerase II showed that the intensity of DNA cleavage and the pattern of cleavage were similar for DHVP, VP-Q and etoposide. In contrast, the VP-OMe failed to induce DNA cleavage, indicating that the presence of 4'-OH is essential for metabolism, induction of topoisomerase II-mediated DNA cleavage and cytotoxicity of etoposide and its analogues.

Breast Neoplasms↗

PubMed1990

Free radicals and anticancer drug resistance: oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors.

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.

Animals↗

PubMed1990

Tyrosinase-induced free radical formation from VP-16,213: relationship to cytotoxicity.

Tyrosinase-dependent activation of hydroxybenzenes forms reactive compounds, including catechols and o-quinones, and some of which show antitumor activity against pigmented melanomas. Since VP-16 is a phenoxy-containing antitumor drug, forms free radicals and reactive o-quinones during peroxidative activation, we evaluated the cytotoxicity of VP-16 to both tyrosinase-containing and non-tyrosinase-containing tumor cells. Our results show that VP-16 is significantly more cytotoxic to B-16/F-10 melanoma cells than human MCF-7 breast tumor cells. Phenylthiocarbamide, an inhibitor of tyrosinase activity, selectively decreased VP-16 toxicity only in melanoma cells. Furthermore, VP-16 was readily activated to its phenoxy free radical intermediate by purified tyrosinase, indicating tyrosinase may play a role in VP-16 toxicity in pigmented melanomas.

Animals↗

PubMed1990

Potentiation of doxorubicin cytotoxicity by (+)-1,2-bis-(3,5-dioxopiperazinyl-1-yl) propane (ICRF-187) in human leukemic HL-60 cells.

The bisdioxopiperazine propane, ICRF-187, has been reported to potentiate doxorubicin cytotoxicity in certain tumor cell lines; however, the mechanism of this interaction is not known. In order to define the mechanism of this interaction, we examined the effects of ICRF-187 on doxorubicin cytotoxicity, free radical formation, and drug accumulation in human leukemia HL-60 cells. Studies show that ICRF-187 synergistically potentiated doxorubicin cytotoxicity in HL-60 cells. This potentiation of doxorubicin cytotoxicity by ICRF-187 appeared to result from enhanced drug dependent free radical formation without effecting doxorubicin uptake in HL-60 cells.

Biological Transport↗

PubMed1990

P-glycoprotein-independent mechanism of resistance to VP-16 in multidrug-resistant tumor cell lines: pharmacokinetic and photoaffinity labeling studies.

The interaction of etoposide (VP-16), Vinca alkaloids, and verapamil with the P-glycoprotein (P-gp) was studied in human breast (MCF-7) and Chinese hamster lung (DC3F) cell lines and the corresponding multidrug-resistant MCF-7/ADR and DC3F/ADX tumor cell lines, selected for resistance to Adriamycin and actinomycin D, respectively, and overexpressing P-gp. Verapamil (10 microM) markedly reversed resistance to vincristine (11-fold in DC3F/ADX and 125-fold in MCF-7/ADR; 1-hr exposure), but it had a very modest effect on resistance to VP-16 (3- to 4-fold; 1-hr exposure). Resistant cells accumulated 2- to 4-fold less VP-16 and vincristine than the parental cell lines. Verapamil (10 microM) significantly increased accumulation and retention of vincristine, but not of VP-16, in resistant cell lines. Photoaffinity labeling of resistant cell lines with radioactive analogs of verapamil [N(p-azido-3-125I-salicyl)-N'-beta-aminoethylverapamil (NASVP)] and vinblastine[N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine (NASV)] showed distinctly labeled P-gp bands in both resistant cell lines, compared with wild-type cells. Excess nonradioactive vinblastine or verapamil effectively competed with the P-gp photolabeling by either NASVP or NASV, with IC50 levels of 0.6 and 10 microM, respectively. In contrast, nonradioactive VP-16 was 100- to 500-fold less potent than vinblastine in competing with P-gp photolabeling, suggesting that VP-16 has significantly lower affinity for P-gp than Vinca alkaloids have. Taken together, our data indicate that P-gp glycoprotein by itself may not be important in the transport/efflux of VP-16 and, thus, in the mechanism of resistance to VP-16 in these cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Anthracyclines.

After twenty years, understanding the mechanisms of tumor cells kill by anthracyclines still remains an active area of research. Of many mechanisms described for this class of drugs, efforts in the last year have focused on defining the role of free radical formation, topoisomerase II-induced DNA breakage, and P-170-dependent cellular accumulation of anthracyclines in tumor cell kill and resistance. First, in a number of tumor cell lines, the formation of free radical species from anthracyclines has been implicated in the cell killing. Modulation of detoxification pathways in a drug-resistant cell line e.g depletion of GSH, a substrate for peroxidase and transferase, enhanced both the formation of oxy-radicals and adriamycin cytotoxicity. It should be noted, however, that these findings are not true for every cell line examined, and free radical-mediated tumor kill may be cell- or tissue-specific. Second, anthracyclines-mediated topo II-dependent DNA cleavage was observed in most cell lines and reduced breaks were found in resistant cells. The decrease in single-strand breaks, however, neither correlated with the degree of resistance nor with differences in the relative topo II activity, which was in most cases only two-fold less in resistant cells than in sensitive cells. Finally, the reduced accumulation of the drug does not appear to be the only contributing factor in multidrug resistant cells and P-170 is not the only protein overexpressed in certain cells, e.g., an 85,000 Da protein may also be linked to adriamycin resistance. Although GST protein is overexpressed in most adriamycin resistant cells along with mdr1 gene, current evidence suggests that this protein may not be directly involved in adriamycin resistance. Taken together, both the mechanism of action and resistance to this class of drug likely vary among cell lines. Clinical studies in the past year have brought about interesting refinements in anthracycline-containing chemotherapy; ICRF-187 (by itself also cytotoxic) seems to offer protection against cardiac toxicity, while implicating iron in the mediation of cardiac damage. Out of a large number of newer anthracycline derivatives, clinical evidence indicates only a modest increase in therapeutic index with a few analogs, perhaps idarubicin and epirubicin. It is not yet clear that being able to receive more milligrams (or more cycles) of anthracycline eventually translates into a significantly better response rate or in a survival advantage. Much less clear is whether patients refractory to adriamycin may derive any benefit from newer anthracyclines.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Synergistic antitumor activity of etoposide and human interleukin-1 alpha against human melanoma cells.

To investigate the possibility of increased activity of cytotoxic anticancer drugs combined with cytokines, we treated human melanoma cells with combinations of etoposide (VP-16) and human recombinant interleukin-1 alpha (rIL-1 alpha). We evaluated the combined cytotoxic effects of VP-16 and rIL-1 alpha using A375-C6 cells, which are sensitive to rIL-1 alpha, and A375-C5 cells, a clonal variant line resistant to rIL-1 alpha. We used the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromid e) assay and the inhibition of [3H]thymidine incorporation into DNA. We analyzed data using the median effects principle of Chou and Talalay (Chou's analysis). The calculated combination index values, at a dose ratio of VP-16 to rIL-1 alpha of 12:1 in simultaneous exposure, indicated synergistic cytotoxicity toward both A375-C6 cells and A375-C5 cells. We observed more pronounced synergism with VP-16 and rIL-1 alpha toward the A375-C5 IL-1 alpha-resistant melanoma cells. These results suggest that rIL-1 alpha combined with cytotoxic antitumor drugs may provide increased benefit in the treatment of malignant melanoma.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1989

Free-radical formation by mitomycin C and its novel analogs in cardiac microsomes and the perfused rat heart.

Using a spin-trapping technique, we have examined free-radical formation by mitomycin C and its analogs, BMY 25282 and BMY 25067, in rat cardiac microsomes and isolated perfused rat hearts. All three drugs stimulated 2--4-fold OH radical formation in cardiac microsomes which was inhibited by SOD and catalase. Superoxide anion radical was also detected in the presence of diethylenetetraaminopentaacetic acid. Addition of DMSO yielded methyl radicals, thus indicating the production of free OH under these conditions. Similar stimulation of OH formation (2--3-fold) in the perfusates from rat hearts was detected with all three drugs. Perfusion with catalase (550 U/ml) completely suppressed the OH signal both in the presence and absence of the drugs, thus suggesting the intermediacy of hydrogen peroxide. However, BMY 25067-induced OH formation was more sensitive to inhibition by superoxide dismutase (SOD) and the iron chelator ICRF-187. Perfusion with DMSO produced methyl radicals at the expense of OH in the presence of all three drugs. SOD and catalase inhibited DMPO-OH signals, indicating that most of the OH formation was extracellular in this setting. While mitomycin C and BMY 25067 (up to 10 microM) did not affect the heart rate, perfusion with 10 microM BMY 25282 caused acute arrhythmia and cardiac standstill within 20 min. An initial surge in OH formation (2-fold) accompanied this cardiotoxic effect. Both the arrhythmia and the free radical signal were partially blocked by SOD, catalase and ICRF-187, indicating that iron-dependent oxygen radical formation from BMY-25282 (and possibly other compounds) is involved, in part, in inducing toxic manifestations in the rat heart and possibly in clinic.

Animals↗

PubMed1989

Adriamycin activation and oxygen free radical formation in human breast tumor cells: protective role of glutathione peroxidase in adriamycin resistance.

Previous studies with Adriamycin-sensitive and -resistant (ADRR) MCF-7 human breast tumor cell lines indicated that Adriamycin formed significantly less hydroxyl radical (.OH) as the result of enhanced detoxification of reactive oxygen intermediates in the ADRR cell line. In order to further define the sites of drug activation and the role of detoxification mechanisms in free radical levels, subcellular fractions were isolated from these two cell lines and free radical formation in the presence of Adriamycin was examined by using electron spin resonance spectroscopy. Studies reported here show that considerable NADPH-cytochrome P-450 reductase and NADH dehydrogenase activities were present in microsomes and mitochondria, respectively, and in nuclei obtained from these cells, and the relative activity of NADH dehydrogenase was 2-fold higher in the mitochondrial fraction of ADRR cells compared to the mitochondrial fraction from the parental wild type cells. In the presence of Adriamycin and a reducing cofactor (NADPH or NADH), Adriamycin semiquinone free radical, superoxide anion, and .OH were detected in all these fractions. Although only a small difference in the relative amount of oxy radical formation was detected in tumor microsomes, both mitochondria and nuclei of ADRR cells showed an overall 2-fold decreased formation of oxy radicals. The formation of the free radicals was significantly inhibited by superoxide dismutase, catalase, and dimethyl sulfoxide, indicating that free .OH generation was both superoxide and hydrogen peroxide dependent. The addition of purified glutathione peroxidase likewise inhibited .OH formation in a dose-dependent fashion. Similarly, when the lysate from ADRR cells, which contains 12- to 14-fold more glutathione peroxidase than Adriamycin-sensitive cells, was added to reaction mixtures containing Adriamycin-sensitive cells and Adriamycin, the .OH formation was diminished. Decreased free radical formation in nuclei and mitochondria, as a result of detoxification of hydrogen peroxide by glutathione peroxidase, may be significant in the protection of ADRR cells from Adriamycin-induced cell killing.

Biotransformation↗

PubMed1989

Characterization of free radicals produced during oxidation of etoposide (VP-16) and its catechol and quinone derivatives. An ESR Study.

Spectroscopic evidence for the radical-mediated metabolism of VP-16, VP-16 catechol, and VP-16 quinone during enzymatic oxidation and autoxidation has been obtained. Autoxidation of the catechol yields the primary semiquinone together with the primary molecular product VP-16 quinone, which subsequently undergoes hydrolytic oxidation to form secondary quinones and semiquinones. Both primary and secondary phenoxyl radicals were detected during peroxidatic oxidation of VP-16. Neither the primary nor the secondary radicals react with DNA at a detectable rate. Evidence for the production of hydroxyl radical during iron-catalyzed oxidation of VP-16 catechol was obtained. These free radical reactions may have implications for the mechanism of antitumor action of VP-16.

Catechols↗

PubMed1989

Resistance of paraquat and adriamycin in human breast tumor cells: role of free radical formation.

Generation and enhanced detoxification of toxic free radicals by glutathione peroxidase and glutathione transferase in human breast tumor cells have been suggested to play an important role in toxicity and in resistance to adriamycin. We have examined the biochemical basis of paraquat-induced free radical formation and the mechanism of resistance to this agent in human breast tumor cell lines. We have also compared the similarities and differences between adriamycin and paraquat in their mode of free radical formation and tumor cell kill. Anaerobic incubation of paraquat resulted in the formation of the paraquat cation radical in both the sensitive and resistant cells which increased with time and was enhanced by NADPH addition. Our studies show that while both adriamycin and paraquat form hydroxyl radicals (.OH) in these cell lines, adriamycin was 2-3 fold better at reducing oxygen. The formation of .OH was inhibited by exogenously added superoxide dismutase and catalase, indicating the involvement of both superoxide anion radical and hydrogen peroxide. In the adriamycin-resistant cell line, less .OH was formed by each of these drugs. While the .OH appeared to be formed outside by both adriamycin and paraquat in the drug-sensitive cells, experiments using chromium oxalate as a spin-broadening agent suggest that the drug-induced .OH formation in the resistant cells is an intracellular event. The adriamycin-resistant cell line was also cross-resistant to paraquat, suggesting a common mechanism of toxicity for both drugs. However, adriamycin was significantly more toxic (4000-times) to the sensitive cells suggesting that either other mechanisms or site-specific free radical formation are also important in biochemical mechanisms of adriamycin toxicity.

Breast Neoplasms↗