In vivo antitumor activity of the bitter melon (Momordica charantia).

The in vivo antitumor activity of a crude extract from the bitter melon (Momordica charantia) was determined. The extract inhibited tumor formation in CBA/H mice which had been given i.p. injections of 1.0 X 10(5) CBA/Dl tumor cells (77% of the untreated mice with tumors versus 33% of the treated mice with tumors after 6 weeks). The extract also inhibited tumor formation in DBA/2 mice which had been given i.p. injections of either 1 X 10(5) P388 tumor cells (0% of untreated mice survived after 30 days versus 40% survival of the treated mice) or 1 X 10(5) L1210 tumor cells (0% survival of untreated mice versus 100% of treated mice after 30 days). The in vivo antitumor effect required both the prior exposure of tumor cells to the extract (2 hr) in vitro and i.p., biweekly injections of the extract into the mice. The optimum dose for tumor inhibition (8 micrograms protein, biweekly, i.p.) was not toxic to mice for at least 45 days of treatment. This same treatment caused a marked enhancement of C3H mouse thymic cell response to concanavalin A in vitro. When compared to the untreated control mice, the bitter melon-injected animals exhibited a 4-fold-higher incorporation of tritiated thymidine into trichloroacetic acid-precipitable material after 48 hr of exposure to 50 micrograms of concanavalin A. Nylon wool-purified spleen cells from these same bitter melon-treated mice exhibited an enhanced mixed lymphocyte reaction when exposed to irradiated P388 stimulator cells (186% of the untreated control mice). These data indicate that in vivo enhancement of immune functions may contribute to the antitumor effects of the bitter melon extract.

Visual transduction in rod outer segments.

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase gamma-subunit.

Synthetic peptides corresponding to various regions of the light-activated guanosine 3',5'-cyclic monophosphate phosphodiesterase (PDE) gamma-subunit (PDE gamma) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDE gamma residues 31-45, inhibited PDE activity and GTPase activity in a dose-dependent manner. The GTPase activity was inhibited by PDE gamma-3 non-competitively. This region of the PDE gamma subunit may be involved in the direct interaction of transducin and PDE alpha beta with PDE gamma.

Protein kinase C alpha and gamma in N/N 1003A rabbit lens epithelial cell differentiation.

PURPOSE: To determine if Protein Kinase C (PKC) plays a role in the initiation of lens epithelial cell differentiation into a lens fiber cell. METHODS: PKCalpha or PKCgamma was overexpressed in N/N 1003A lens epithelial cells for up to 7 days. Phase contrast microscopy was used to observe morphological changes associated with PKCalpha or PKCgamma overexpression. Cell cycle changes in cells overexpressing PKCalpha or PKCgamma were measured using acridine orange staining and flow cytometry. Crystallin levels in cells overexpressing PKCalpha or PKCgamma were measured using Western blots and RT-PCR. RESULTS: Significant differences in cell cycling were observed between untransfected cells and those overexpressing PKCalpha or PKCgamma. Overexpression of PKCalpha and PKCgamma caused the cells to lose their epithelial-like appearance and elongate. alphaB-crystallin expression was detected in all the samples while alphaA-crystallin was detected only in cells after 7 days of PKCalpha or PKCgamma overexpression. CONCLUSIONS: The observations that alphaA-crystallin is only found in N/N 1003A cells overexpressing PKCalpha or PKCgamma for 7 days along with the finding that a block in the G0/G1 phase of the cell cycle and the consequent morphological changes are observed, indicate that PKCalpha and PKCgamma may have a role in the initiation of differentiation in lens epithelial cells.

PKCalpha and PKCgamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line.

PURPOSE: The overexpression of PKCalpha or PKCgamma for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for alphaA-, alphaB-, beta-, and gamma-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development. METHODS: Cells overexpressing PKCalpha or PKCgamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCalpha or PKCgamma antibodies along with either alphaA-, alphaB-, beta-, or gamma-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either alphaA-, alphaB-, beta-, or gamma-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 microg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis. RESULTS: alphaB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCalpha and PKCgamma overexpression. alphaA-crystallin was only detectable in lentoid bodies after PKCalpha or PKCgamma overexpression. RT-PCR was able to detect beta-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of beta-crystallin protein. No gamma-crystallin expression was noted in these lentoid bodies. CONCLUSIONS: Overexpression of PKCalpha or PKCgamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only alphaB-crystallin but alphaA- and beta-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell.

PKC-gamma phosphorylation of connexin 46 in the lens cortex.

PURPOSE: To identify the role of PKC-gamma in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex. METHODS: The association between PKC-gamma and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors. RESULTS: Co-localization of PKC-gamma and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-gamma was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-alpha was not found in lens cortex or nuclear regions. PKC-gamma could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 microM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-alpha pseudosubstrate inhibitor was not observed. CONCLUSIONS: PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is suggested.

Synthesis and in-vitro cytotoxic activity of phenyl-amino-4-(p-toluenesulfinyl)-trans-1,5-hexadiene.

A novel anticancer drug, 1-phenyl-3-phenylamino-4-(p-toluenesulfinyl-trans-1,5-hexadiene has been synthesized and found to have in-vitro cytotoxicity against P388 (LD50 = 15 micrograms/ml) and L1210 (LD50 = 19 micrograms/ml) murine leukemia cells in culture. The LD50 compared favorably with that for doxorubicin. The compound was more cytotoxic to P388 tumor cells than to normal mouse splenocytes. The compound inhibited the uptake of both tritiated thymidine (42% inhibition) and tritiated uridine (24% inhibition) after 3 h of incubation when used at 5 micrograms/ml. No effect on uptake of tritiated leucine was observed during this time period. The compound was cytotoxic to normal mouse splenocytes which had been stimulated to divide by the mitogen concanavalin A. No effect was found on normal, non-dividing splenocytes. These results suggest that this novel compound is cytotoxic to leukemic cells or other rapidly dividing cells through inhibition of DNA and/or RNA synthesis.

Antibody indications of secondary and superimposed retinal hypersensitivity in retinitis pigmentosa.

Antibody reactions with recognized retinopathy-inducing retinal antigens may be interpreted to reflect ongoing autoimmune events responsible for some forms of vision loss. We sought evidence of secondary and superimposed retinal hypersensitivity indicated by such antibody reactivity in a random group of patients with retinitis pigmentosa. We identified patterns of immunologic reactivity within members of a group of 52 patients with retinitis pigmentosa, which suggests some patients with retinitis pigmentosa may experience consequential superimposed retinal hypersensitivity. Identifying subgroups of patients with retinitis pigmentosa who exhibit indications of retinal hypersensitivity to known uveitopathogenic retinal proteins may permit the reduction of their rate of retinal degradation by immunomodulation.

The cytotoxic and cytostatic effects of the bitter melon (Momordica charantia) on human lymphocytes.

We have previously reported that a crude extract from the bitter melon (Momordica charantia) killed human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocyte cells at these same doses (Takemoto et al., 1980). We now report that the crude preparation has both cytostatic and cytotoxic activities which are heat stable and trypsin-sensitive. Time and dose-response curves suggest that the factors act quickly, perhaps by entry into the cell. The effects of the crude extract are complete after only 2 hr of exposure. These activities are not due to the presence of the lectins from bitter melon seeds, as these purified proteins had no activity against human lymphocytic cells.

Effect of trichosanthin an anti-leukemia protein on normal mouse spleen cells.

Trichosanthin, from Trichosanthes kirilowii is a member of the Cucurbitaceae family. It has been reported to have anti-cancer activity. The effects of Trichosanthin peptides (15 amino acids in length) on ConA stimulated incorporation of 3[H] thymidine into cultured, primary mouse spleen cells and L1210 cells were determined. Both spleen cells and L1210 cells were plated at 7.5 x 10(5) cells/ml in a microtiter plate. Different concentrations of the peptide (0.5, 5, 25, and 50 micrograms/ml) and ConA (1 microgram/ml) were added with serum free media, and half of the samples were labeled with 1 microCi/well of 3[H] thymidine. The additional half of the cell samples were used to determine cell number and % viability. After 24 hours incubation, the N-terminal peptides (amino acid residues #1-15 and 16-30) caused increases in ConA stimulated incorporation of 3[H] thymidine into normal spleen cells at low concentrations of the peptides (5 micrograms/ml). The viability of spleen cells and L1210 cells were not affected by these peptides at 5 micrograms/ml. These N-terminal peptides (#1-15 and 16-30) were tested for in vivo anti-tumor activity. There was a delay of tumor formation in the treated vs control group. The results suggest that N-terminal sequences of trichosanthin have anti-tumor activity.

Anticancer activities of 2,5,8,9-substituted 6-oxo-1,2,3,4,5,6-hexahydrophenanthridines on multi-drug-resistant phenotype cells.

Lycorine is a member of the alkaloid group from the bulb of the amaryllidaceae. This drug has been reported to have anticancer activity. Several synthetic intermediates obtained during the synthetic study of anticancer drugs based on the lycorine structure, were tested for anticancer activity using three cell lines: L1210 and HL60 cell lines which were resistant (R) or sensitive (S) to adriamycin. The two synthetic intermediates, 2-acetoxy- and 2-hydroxy-5-allyl-8,9-methylenedioxy-6-oxo-1,2,3,4,5,6-hexahydr ophenanthridine (1 and 2), both had anticancer activity in all three cell lines. However, the LD50 for the precursors was about 20 fold greater than for the native lycorine. Both 1 and 2 were cytotoxic to the adriamycin-resistant cell line, indicating that these drugs are not affected by the multidrug resistance factors. When low doses of the compounds were used, the HL60R cell line could be induced to differentiate to a cell which expressed a macrophage specific protein. These results suggest that phenanthridines 1 and 2 can be used on cells which are resistant to adriamycin, and that one mechanism of action is the induction of differentiation.

Decreases in Raf-1 levels in galactosaemic lens epithelial cells are partially reversed by myo-inositol.

Changes in the amounts and localization of protein kinase C (PKC) isoforms occur in galactosaemic lens epithelial cells. A link between PKC changes and myo-inositol depletion has been suggested. Raf-1, a component of a Ras pathway, is a substrate for PKC. Raf-1 levels were measured in galactosaemic lens epithelial cells grown with or without myo-inositol. Raf-1 levels were measured by densitometric scanning of Western blots from cells grown with or without 40 mmol/l galactose or 40 mmol/l galactose plus 1.0 micromol/l myo-inositol for 1, 3, 5 or 7 days. Scans were compared to those for PKCalpha, an isoform of PKC and to 14-3-3, a protein which binds to Raf-1. Cell growth was quantitated by thymidine incorporation. Raf-1 levels were decreased in bovine lens epithelial cells after 3, 5 or 7 days (33% of control) of growth in 40 mmol/l galactose. Addition of 1 micromol/l myo-inositol reversed this decrease at day 3, but not after 5 or 7 days of growth in 40 mmol/l galactose. PKCalpha and 14-3-3 levels were not affected by galactose. The decrease in Raf-1 was not a result of cell growth as measured by thymidine incorporation. These results suggest that Raf-1 levels are decreased during galactosaemia. This was only partially reversed by the addition of myoinositol.

Protein kinase C in galactosemic and tolrestat-treated lens epithelial cells.

Using isozyme-specific anti-peptide antisera against peptides from the alpha-, beta-, gamma-, delta-, epsilon-, and zeta-isoforms of brain protein kinase C (PKC), we have identified proteins in bovine lens epithelial cells, in culture, that were reactive with these antisera. Western blots of lens epithelial cell homogenates showed that PKC-alpha antisera reacted with a major protein, and PKC-gamma antisera reacted with a minor protein. When the lens epithelial cells were cultured in media supplemented with 40 mM galactose, to model the conditions of sugar cataracts, a decrease in PKC-gamma, but not in PKC-alpha was observed. These were normalized if the cells were cultured in 40 mM galactose media supplemented with an inhibitor of aldose reductase, Tolrestat (10 microM). These results suggest that changes in PKC isoforms occur in the galactosemic diabetic state.

Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry.

The lens epithelial cells are a primary site of involvement in galactosemia. Changes in their size, shape and proliferative capacity have been observed upon exposure to high galactose. In this report, changes in the cell cycle pattern of normal and galactosemic lens epithelial cells were examined by use of flow cytometry. Both changes in DNA and RNA were observed using the fluorochrome, acridine orange. Under the appropriate conditions acridine orange can be used to differentiate double-stranded DNA from single-stranded RNA. Using this approach, the DNA and RNA of normal and galactosemic (1, 4, or 7 days) lens epithelial cells can be compared. The results indicate that lens epithelial cells, when exposed to 40 mM galactose media or 30 mM glucose for 7 days, are induced to enter mitosis. Mannitol did not mimic these results. Changes in the cell cycle pattern were not observed when the cells were treated for 1 or 4 days. Although higher numbers of cells in mitosis were observed after 7 days exposure to 40 mM galactose, a correlation between proliferation, as measured by 3H-thymidine uptake, and mitosis was not possible. Apoptosis was evaluated as a possible explanation for these results. The changes in the DNA staining pattern could be use to monitor lens epithelial cells during galactosemia.

Identification of the gamma-subunit interaction sites in the retinal cyclic-GMP phosphodiesterase beta-subunit.

Using synthetic peptides, the identification of the retinal cyclic-GMP phosphodiesterase (cGMP PDE) interaction sites for the inhibitory gamma-subunit in the catalytic alpha-subunit were recently localized to residues #16-30 and 78-90 in the alpha-subunit (1). In this study, a binding radioimmunoassay (RIA) showed a weak interaction between PDE gamma and PDE beta subunits in PDE beta residues #15-34, and stronger interaction sites were found in residues #91-110 and 211-230. Sequence comparison between PDE alpha and PDE beta illustrate some differences in these regions, particularly in PDE alpha 16-30 and PDE beta 15-34 regions. Differences in interaction sites in PDE alpha and PDE beta for PDE gamma may account for the differences in affinities observed between PDE gamma and the catalytic subunits.

Carriers of the mouse rd gene have reduced levels of the beta subunit of the retinal cyclic GMP phosphodiesterase.

Polyclonal antipeptide antisera have been utilized to quantitate the amount of retinal rod outer segment cGMP phosphodiesterase alpha and beta catalytic subunits present in retinas from C57BL/6J mice which are normal or carriers for the rd gene defect. Results suggest that the quantity of PDE-beta subunit is reduced in carrier mice while PDE-alpha and PDE-gamma are not affected. In 21-day-old mice, the PDE-beta was reduced by about one-half while adult carrier mice had even more reduced levels of PDE-beta. Since PDE alpha was not reduced, this suggests that synthesis of PDE alpha and PDE beta may not be coordinately controlled.

The effect of the gamma-subunit of the cyclic GMP phosphodiesterase of bovine and frog (Rana catesbiana) retinal rod outer segments on the kinetic parameters of the enzyme.

Rod-outer-segment cyclic GMP phosphodiesterase (PDE) (subunit composition alpha beta gamma 2) contains catalytic activity in alpha beta. The gamma-subunits are inhibitors. Removal of the gamma-subunits increases Vmax. without affecting the Km. The inhibitory effect of a single gamma-subunit (alpha beta gamma) on the Vmax. of alpha beta is much greater in bovine than in frog (Rana catesbiana) PDE. Bovine PDE in the alpha beta gamma 2 state has a Vmax. that is 2.6 +/- 0.4% of the Vmax. of alpha beta. The removal of one gamma-subunit to give alpha beta gamma results in a Vmax. 5.2 +/- 1% of that for maximal activity. Frog alpha beta gamma 2 has a Vmax. 10.8 +/- 2%, and alpha beta gamma has a Vmax. 50 +/- 18%, of the Vmax. of alpha beta. These data suggest that a single gamma-subunit can inhibit the catalytic activity of active sites on both alpha- and beta-subunits in bovine, but not in frog, rod-outer-segment PDE.

Purification and characterization of a cytostatic factor from the bitter melon Momordica charantia.

In this report we describe the purification and characterization of a cytostatic factor from the bitter melon (Momordica charantia). As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with an apparent molecular weight of 11,000. The factor is not sensitive to boiling or to pretreatments with trypsin, ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake studies, the purified factor preferentially inhibits RNA synthesis in intact tissue culture cells. Some inhibition of protein synthesis and DNA synthesis also occurs. The factor is preferentially cytostatic for IM9 human leukemic lymphocytes when compared to normal human peripheral blood lymphocytes.