Radiosensitizer Effect of ß-Apopicropodophyllin against Colorectal Cancer via Induction of Reactive Oxygen Species and Apoptosis.

ß-apopicropodophyllin (APP), a derivative of podophyllotoxin (PPT), has been identified as a potential anti-cancer drug. This study tested whether APP acts as an anti-cancer drug and can sensitize colorectal cancer (CRC) cells to radiation treatment. APP exerted an anti-cancer effect against the CRC cell lines HCT116, DLD-1, SW480, and COLO320DM, with IC50 values of 7.88 nM, 8.22 nM, 9.84 nM, and 7.757 nM, respectively, for the induction of DNA damage. Clonogenic and cell counting assays indicated that the combined treatment of APP and γ-ionizing radiation (IR) showed greater retardation of cell growth than either treatment alone, suggesting that APP sensitized CRC cells to IR. Annexin V-propidium iodide (PI) assays and immunoblot analysis showed that the combined treatment of APP and IR increased apoptosis in CRC cells compared with either APP or IR alone. Results obtained from the xenograft experiments also indicated that the combination of APP and IR enhanced apoptosis in the in vivo animal model. Apoptosis induction by the combined treatment of APP and IR resulted from reactive oxygen species (ROS). Inhibition of ROS by N-acetylcysteine (NAC) restored cell viability and decreased the induction of apoptosis by APP and IR in CRC cells. Taken together, these results indicate that a combined treatment of APP and IR might promote apoptosis by inducing ROS in CRC cells.

ß-Apopicropodophyllin functions as a radiosensitizer targeting ER stress in non-small cell lung cancer.

AIMS: In this study, we examined whether ß-apopicropodophyllin (APP) could act as a radiosensitizer in non-small cell lung cancer (NSCLC) cells. MAIN METHODS: The in vitro radiosensitizing activity of APP was demonstrated with clonogenic assay, immunoblotting, Annexin V-Propidium iodide (PI) assay, BrdU incorporation, detection of mitochondrial ROS/intracellular of H2O2, mitochondrial membrane potential detection, and performing of isolation of mitochondrial and cytosolic fractions. The in vivo radiosensitizing activity of APP was determined in xenografted mice with co-treatment of APP and IR based on measurement of tumor volumes and apoptotic cell death. KEY FINDINGS: The results of a clonogenic assay indicated that a combination of APP and γ-ionizing radiation (IR) inhibits cell growth and increases cell death in NSCLC cells. Several signal transduction pathways were examined for their potential involvement in the apparent radiosensitization effect of APP, as assessed by immunoblotting analyses and mitochondrial potential determination in vitro. Treatment of NCI-H460 cells with 15 nM APP and NCI-H1299 cells with 10 nM APP yielded dose-enhancement ratios of 1.44 and 1.24, respectively. Enhanced ER stress, disrupted mitochondrial membrane potential, and increased reactive oxygen species (ROS) were observed in cells co-treated with APP and IR, and this was followed by the cytosolic release of cytochrome c and consequent activation of caspase-3 and -9. Notably, inhibition of JNK, which prevents caspase activation, blocked the APP/IR-induced activations of ER stress and apoptotic cell death. In NCI-H460 or NCI-H1299 cell-xenografted mice, APP/IR treatment delayed the time it took tumors to reach a threshold size by 22.38 and 16.83 days, respectively, compared with controls, to yield enhancement factors of 1.53 and 1.38, respectively. SIGNIFICANCE: APP has a radiosensitizing function derived from its ability to induce apoptotic cell death via activation of ER stress, disruption of mitochondrial membrane potential, and induction of the caspase pathway.

A novel anti-cancer role of ß-apopicropodophyllin against non-small cell lung cancer cells.

We previously reported that podophyllotoxin acetate (PA) inhibits the growth and proliferation of non-small cell lung cancer (NSCLC) cells and also makes them more sensitive to radiation and chemotherapeutic agents. In an attempt to enhance PA activity, we synthesized 34 derivatives based on podophyllotoxin (PPT). Screening of the derivative compounds for anti-cancer activity against NSCLC led to the identification of ß-apopicropodophyllin (APP) as a strong anti-cancer agent. In addition to its role as an immunosuppressive regulator of the T-cell mediated immune response, the compound additionally showed anti-cancer activity against A549, NCI-H1299 and NCI-460 cell lines with IC50 values of 16.9, 13.1 and 17.1 nM, respectively. The intracellular mechanisms underlying the effects of APP were additionally examined. APP treatment caused disruption of microtubule polymerization and DNA damage, which led to cell cycle arrest, as evident from accumulation of phospho-CHK2, p21, and phospho-Cdc2. Moreover, APP stimulated the pro-apoptotic ER stress signaling pathway, indicated by elevated levels of BiP, phospho-PERK, phospho-eIF2α, CHOP and ATF4. We further observed activation of caspase-3, -8 and -9, providing evidence that both intrinsic and extrinsic apoptotic pathways were triggered. In vivo, APP inhibited tumor growth of NSCLC xenografts in nude mice by promoting apoptosis. Our results collectively support a novel role of APP as an anticancer agent that evokes apoptosis by inducing microtubule disruption, DNA damage, cell cycle arrest and ER stress.

Glucagon and/or IGF-1 Production Regulates Resetting of the Liver Circadian Clock in Response to a Protein or Amino Acid-only Diet.

The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment.

HOTAIR-mediated reciprocal regulation of EZH2 and DNMT1 contribute to polyphyllin I-inhibited growth of castration-resistant prostate cancer cells in vitro and in vivo.

BACKGROUND: Polyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been reported to exhibit antitumor effects. However, the detailed molecular mechanism underlying this has not been elucidated. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Flow cytometry assays, respectively. Cell invasion and migration were examined by Transwell invasion and wound healing assays. Western blot analysis was performed to examine the protein expressions of zeste homolog 2 (EZH2), DNA methyltransferase 1 (DNMT1). QRT-PCR was used to examine the levels of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR). Small interfering RNAs (siRNAs) method was used to knockdown HOTAIR. Exogenously expressions of HOTAIR, DNMT1 and EZH2 were carried out by Transient transfection assays. EZH2 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro. RESULTS: We showed that PPI significantly inhibited growth, induced cell cycle arrest of castration-resistant prostate cancer (CRPC) cells. In addition, PPI also reduced the migration and invasion in CRPC cells. In mechanism, we found that PPI decreased the protein expressions of EZH2, DNMT1 and levels of HOTAIR. Interestingly, silenced HOTAIR reduced EZH2 and DNMT1 protein expressions. On the contrary, exogenously expressed HOTAIR resisted PPI-inhibited EZH2 and DNMT1 protein expressions, EZH2 promoter activity and cell growth. Moreover, excessive EZH2 antagonized PPI-suppressed DNMT1 protein expression or vice versa. Consistent with this, PPI inhibited tumor growth, HOTAIR, the protein expressions of DNMT1 and EZH2 in vivo. CONCLUSION: Our results show that PPI inhibits growth of CRPC cells through inhibition of HOTAIR expression, subsequently; this results in the repression of DNMT1 and EZH2 expressions. The interactions among HOTAIR, DNMT1 and EZH2, and reciprocal regulation of DNMT1 and EZH2 contribute to the overall responses of PPI. This study reveals a novel mechanism for HOTAIR-mediated regulating DNMT1 and EZH2 in response to PPI in inhibition of the growth of CRPC cells.

Structure-activity relationship of chemical defenses from the freshwater plant Micranthemum umbrosum.

Vascular plants produce a variety of molecules of phenylpropanoid biosynthetic origin, including lignoids. Recent investigations indicated that in freshwater plants, some of these natural products function as chemical defenses against generalist consumers such as crayfish. Certain structural features are shared among several of these anti-herbivore compounds, including phenolic, methoxy, methylenedioxy, and lactone functional groups. To test the relative importance of various functional groups in contributing to the feeding deterrence of phenylpropanoid-based natural products, we compared the feeding behavior of crayfish offered artificial diets containing analogs of elemicin (1) and beta-apopicropodophyllin (2), chemical defenses of the freshwater macrophyte Micranthemum umbrosum. Both allyl and methoxy moieties of 1 contributed to feeding deterrence. Disruption of the lactone moiety of 2 reduced its deterrence. Finally, feeding assays testing effects of 1 and 2 at multiple concentrations established that these two natural products interact additively in deterring crayfish feeding.

Roles of NF-kappaB and SP-1 in oxidative stress-mediated induction of platelet-derived growth factor-B by TNFalpha in human endothelial cells.

Platelet-derived growth factor-B (PDGF-B) is upregulated by proinflamatory stimuli in the early stages of atherosclerosis. However, its mechanisms are not fully elucidated. In the present study, by using the antioxidant N-acetylcysteine (NAC), we investigated in human umbilical vein endothelial cells (HUVECs) the roles of oxidative stress in PDGF-B expression induced by tumor necrosis factor alpha (TNFalpha) and its underlying mechanisms. Exposure of HUVECs to TNFalpha (200 U/ml) for 24 hours caused significant increases of both the PDGF-B expression and its promoter/enhancer activity, which were abolished by NAC (20 mmol/L). Accordingly, a prolonged oxidative stress was induced by TNFalpha and that was prevented by pretreatment with NAC. Electrophoresis mobility shift assay (EMSA) and Western blot analysis showed that both the nuclear factor-kappaB (NF-kappaB) and the specificity protein-1 (SP-1) were activated by TNFalpha. However, NAC only partially inhibited the TNFalpha-induced activation of NF-kappaB, but abolished the activation of SP-1. Mutation of the NF-kappaB binding site resulted in a moderate reduction in the TNFalpha-induced activity of PDGF-B promoter/enhancer, whereas mutation of SP-1 binding site resulted in an absence of induction by TNFalpha. These results suggest that oxidative stress mediates the TNFalpha-induced expression of PDGF-B in HUVECs through redox-sensitive transcription factors, predominantly the SP-1 and possibly, to some extent of NF-kappaB.

Potato disc tumor induction assay: a multiple mode of drug action assay.

The study reported herein utilized the Agrobacterium tumefaciens-induced potato disc tumor assay. The objective was to verify the detection of antineoplastic activity in the potato disc tumor induction assay, regardless of the mode of antineoplastic drug action. Camptothecin, paclitaxel, podophyllin, vinblastine and vincristine were tested, each with a different mode of action. All drugs tested inhibited tumor induction. The order of activity was: camptothecin = paclitaxel = vinblastine < podophyllin = vincristine. No effect on the viability of the bacterium was detected. The A. tumefaciens-induced potato disc tumor assay was an effective indicator of antitumor activity regardless of the mechanism of drug action. Thus, this assay would be acceptable as a primary general screen for antineoplastic activity of various crude extracts, as well as for purified fractions, regardless of mode of inhibitory action on tumor formation.

Antioxidative activity of spin labeled derivatives of podophyllic acid hydrazide.

AIM: To study the relationship between structure and antioxidation activity of spin labeled derivatives of podophyllic acid hydrazide (GP) in tissues and red blood cells (RBC) from rats. METHODS: The homogenate of liver, heart, and kidneys of rats was used to measure malondialdehyde (MDA) spontaneous generated and induced by hydroxyl free radical generation system (Fe2+-ascorbic acid, FRGS) or doxorubicin (DOX) by TBA colorimetric method. H2O2-caused hemolysis was determined spectrophotometrically. Superoxide anion from zymosan-stimulated neutrophils of rats was evaluated by NBT-reduction assay. RESULTS: GP1 and GP1OH obviously inhibited MDA formation either spontaneously or induced by FRGS and DOX and antagonized hemolysis induced by H2O2, but GP and GP1H showed less potent activity. GP1 also inhibited the formation of superoxide anion from activated neutrophils of rats. CONCLUSION: Introduction of nitroxyl radical moieties into GP generated potent derivatives with antioxidative activity. The essential antioxidation active groups of spin labeled derivative of GP are NO or NOH group in nitroxyl radical moieties.

Oral hairy leukoplakia: an Epstein-Barr virus-associated disease of patients with HIV.

Oral hairy leukoplakia is a common, benign, opportunistic EBV infection of the oral cavity of patients with HIV. It is important to differentiate hairy leukoplakia from other, more serious, oral lesions that may have a similar clinical appearance. In some cases, this is best accomplished by biopsy and histologic examination of the tissue. Several treatment options are available for symptomatic hairy leukoplakia lesions, but none prevent the recurrence of the lesion after therapy. Research studies into the pathogenesis and treatment of oral hairy leukoplakia and other HIV-associated and EBV-associated oral lesions are currently being conducted at the Bering Dental Clinic in Houston.

Antitumor activity of a new low immunosuppressive derivative of podophyllotoxin (GP-11) and its mechanisms.

The spin-labeled derivative of podophyllotoxin, N'-podophyllic acid-N-[3-(2,2,5,5-tetramethyl pyrrolinenyloxy)] semicarbazide (GP-11), was synthesized and tested for its antitumor activity against mouse transplantable tumors, Sarcoma-180, Hepatoma-A, P388 leukemia and Ehrlich ascites carcinoma. At an equitoxic dose, the antitumor activity of GP-11 was similar to that of etoposide (VP-16). However, the immunosuppressive effects of GP-11 were weaker than that of VP-16. In vitro, GP-11 and VP-16 inhibited the proliferation of human lymphoid leukemia Molt 4B cells and suppressed DNA and protein syntheses, but the effect of GP-11 and VP-16 on cell cycle progression was different. The mitotic index was increased by GP-11 and reduced by VP-16. On the basis of flow cytometric bromodeoxyuridine (BrdU)/DNA analysis, GP-11 and VP-16 resulted in the accumulation of cells in the S and G2/M phases. G2/M arrest by GP-11 on cell cycle progression was stronger than that of VP-16, while S arrest was weaker than that of VP-16. After the removal of drugs, the arrest by GP-11 and VP-16 still existed and was irreversible. These results may provide insights into the structure-activity relationships and the design of novel derivatives of podophyllotoxin useful in cancer chemotherapy.

A podofilina é a melhor forma de tratar o condiloma acuminado? / Is the podophyllin the better therapy to condylomata acuminata?

Os autores realizam uma revisäo das informaçöes atuais e já consolidadas a respeito do fármaco, mostrando suas propriedades farmacodinâmicas e farmacocinéticas, suas indicaçöes e problemas associados e, completando, informaçöes de interesse do médico generalista e do paciente na aplicaçäo fora do consultório. Por fim, säo apresentadas opçöes terapêuticas para o tratamento de sua principal indicaçäo, o condiloma acuminado.

Stereoselective reactions. XVIII. Synthesis and cytotoxicity of the demethyl derivatives of steganes.

Demethyl derivatives of steganes and deoxypodorhizon, 3, 4, 6, 7, 9, 10, 12, 13, 18, 23, were prepared by the selective demethylation of the methoxy group of steganes and deoxypodorhizon, 2, 5, 8, 11, 22. The cytotoxicity of these derivatives was evaluated against KB cell and was found not to exceed that of the parent steganes. 4-Demethyldeoxypodorhizon (18) was found to show more potent cytotoxicity than deoxypodorhizon.

Structure-activity relationships of podophyllin congeners that inhibit topoisomerase II.

Various analogs of etoposide have been studied and compared in different tests in order to identify which tests best correlate with antitumor activity. These tests included DNA breakage assays using standard alkaline elution procedures as a means of studying topoisomerase II inhibition in intact cells, cytotoxicity studies in naturally sensitive and resistant human carcinoma cell lines, in vitro assays of the effect of the different congeners on topoisomerase II activity, and a preliminary evaluation of the ability of etoposide and teniposide to induce resistance. As in previous studies, a direct correlation was seen between double strand DNA breakage and cytotoxicity but not between single strand DNA breakage and cytotoxicity. Analogs with blocked 4'-hydroxyl groups were poor antitumor agents but were still capable of inhibiting topoisomerase II as evidenced by the production of DNA breaks. However, this DNA breakage was qualitatively different from that produced by VP16. None of the analogs were able to overcome either naive or acquired drug resistance. The dihydroxy analog of VP16, a possible bioactivated analog, was much less potent and possibly less stable than VP16. A model is proposed for the inhibition of topoisomerase II by demethylepipodophyllotoxins that may explain the relationship between double strand DNA breakage and cytotoxicity.

Oxidized sterols inhibit the formation of podophyllin-induced metaphase figures in mouse vaginal epithelia.

The antimitotic activity of oxidized derivatives of cholesterol was investigated using an assay developed by Van Scott and Bonder. In this assay, a drug that has antimitotic activity and is not a metaphase-blocking agent will inhibit the formation of podophyllin-induced metaphase figures, as counted on histologic specimens. Mouse vaginal epithelia were classified as being estrogen or progesterone predominant on the basis of histologic criteria. Podophyllin-injected mice in the estrogenic phase of the estrus cycle demonstrated high metaphase-figure counts, with an average of 284.86 +/- 132.01. In this group, all intravaginally administered compounds, inhibited the formation of metaphase figures, including a propylene-glycol ethanol vehicle (60% suppression); thus, it is concluded that animals in this phase are not a suitable model for assaying antimitotic activity. Mice in the progesterone-predominant phase of the estrus cycle had lower counts of podophyllin-induced metaphase figures, i.e., 142.13 +/- 39.29. In this group, 25-OH-cholesterol was the most effective inhibitor (59% suppression), followed by 7-ketocholesterol (48% suppression) and methotrexate (40% suppression). Cholesterol (5% suppression) and vehicle (20% suppression) did not have any significant effects. Progesterone-predominant epithelium was only susceptible to methotrexate and oxidized sterols. This suggests that oxidized sterols may have antimitotic activity.