Structure-Based Optimization of Carbendazim-Derived Tubulin Polymerization Inhibitors through Alchemical Free Energy Calculations.

Carbendazim derivatives, commonly used as antiparasitic drugs, have shown potential as anticancer agents due to their ability to induce cell cycle arrest and apoptosis in human cancer cells by inhibiting tubulin polymerization. Crystallographic structures of α/ß-tubulin multimers complexed with nocodazole and mebendazole, two carbendazim derivatives with potent anticancer activity, highlighted the possibility of designing compounds that occupy both benzimidazole- and colchicine-binding sites. In addition, previous studies have demonstrated that the incorporation of a phenoxy group at position 5/6 of carbendazim increases the antiproliferative activity in cancer cell lines. Despite the significant progress made in identifying new tubulin-targeting anticancer compounds, further modifications are needed to enhance their potency and safety. In this study, we explored the impact of modifying the phenoxy substitution pattern on antiproliferative activity. Alchemical free energy calculations were used to predict the binding free energy difference upon ligand modification and define the most viable path for structure optimization. Based on these calculations, seven compounds were synthesized and evaluated against lung and colon cancer cell lines. Our results showed that compound 5a, which incorporates an α-naphthyloxy substitution, exhibits the highest antiproliferative activity against both cancer lines (SK-LU-1 and SW620, IC50 < 100 nM) and induces morphological changes in the cells associated with mitotic arrest and mitotic catastrophe. Nevertheless, the tubulin polymerization assay showed that 5a has a lower inhibitory potency than nocodazole. Molecular dynamics simulations suggested that this low antitubulin activity could be associated with the loss of the key H-bond interaction with V236. This study provides insights into the design of novel carbendazim derivatives with anticancer activity.

Exploring the Influence of Spacers in EDTA-ß-Cyclodextrin Dendrimers: Physicochemical Properties and In Vitro Biological Behavior.

The synthesis of a new family of ethylenediaminetetraacetic acid (EDTA) core dimers and G0 dendrimers end-capped with two and four ß-cyclodextrin (ßCD) moieties was performed by click-chemistry conjugation, varying the spacers attached to the core. The structure analyses were achieved in DMSO-d6 and the self-inclusion process was studied in D2O by 1H-NMR spectroscopy for all platforms. It was demonstrated that the interaction with adamantane carboxylic acid (AdCOOH) results in a guest-induced shift of the self-inclusion effect, demonstrating the full host ability of the ßCD units in these new platforms without any influence of the spacer. The results of the quantitative size and water solubility measurements demonstrated the equivalence between the novel EDTA-ßCD platforms and the classical PAMAM-ßCD dendrimer. Finally, we determined the toxicity for all EDTA-ßCD platforms in four different cell lines: two human breast cancer cells (MCF-7 and MDA-MB-231), human cervical adenocarcinoma cancer cells (HeLa), and human lung adenocarcinoma cells (SK-LU-1). The new EDTA-ßCD carriers did not present any cytotoxicity in the tested cell lines, which showed that these new classes of platforms are promising candidates for drug delivery.

Proteomic Profiling Reveals the Induction of UPR in Addition to DNA Damage Response in HeLa Cells Treated With the Thiazolo[5,4-b]Quinoline Derivative D3ClP.

9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinolone (D3ClP) is a bioisostere of N-(4-(acridin-9-ylamino)-3-methoxyphenyl)methanesulfonamide (m-AMSA) a DNA topoisomerase II inhibitor with proven cytotoxic activity and known to induce DNA damage and apoptotic cell death in K562 cells. However, recent evidence is not consistent with DNA topoisomerase II (DNA TOP2) as the primary target of D3ClP, in contrast to m-AMSA. We provide evidence of histone γH2AX phosphorylation at Ser135 in HeLa cells treated with D3ClP, a marker of DNA double strand repair through Mre11-Rad50-Nbs1 (MRN) pathway. Using two-dimensional gel electrophoresis and mass spectrometry, the upregulation of the protein GRP78, the cleavage of Cytokeratin 18, and the downregulation of prothymosine, calumenin, and the α chain of the nascent polypeptide associated complex were observed in HeLa cells treated with D3ClP. An increase in GRP78 has been related with the onset and progression of the unfolded protein response (UPR), a process aimed to reduce endoplasmic reticulum (ER) stress and protein misfolding. The IRE1-α dependent splicing of mRNA encoding X-box binding protein 1 was detected. Microtubule-associated Proteins 1A/1B, Light Chain 3-II (LC3b-II) accumulation was observed, and suggest some involvement of autophagy. The production of the pro-apoptotic protein DNA-damage-inducible protein 153 (GADD-153) was also detected. These results, are consistent with the induction of the UPR and the DNA-Damage Response in D3ClP-treated HeLa cells, and are also consistent with a concurrent apoptotic cell death. J. Cell. Biochem. 118: 1164-1173, 2017. © 2016 Wiley Periodicals, Inc.

Synthesis and Biological Evaluation of 2H-Indazole Derivatives: Towards Antimicrobial and Anti-Inflammatory Dual Agents.

Indazole is considered a very important scaffold in medicinal chemistry. It is commonly found in compounds with diverse biological activities, e.g., antimicrobial and anti-inflammatory agents. Considering that infectious diseases are associated to an inflammatory response, we designed a set of 2H-indazole derivatives by hybridization of cyclic systems commonly found in antimicrobial and anti-inflammatory compounds. The derivatives were synthesized and tested against selected intestinal and vaginal pathogens, including the protozoa Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis; the bacteria Escherichia coli and Salmonella enterica serovar Typhi; and the yeasts Candida albicans and Candida glabrata. Biological evaluations revealed that synthesized compounds have antiprotozoal activity and, in most cases, are more potent than the reference drug metronidazole, e.g., compound 18 is 12.8 times more active than metronidazole against G. intestinalis. Furthermore, two 2,3-diphenyl-2H-indazole derivatives (18 and 23) showed in vitro growth inhibition against Candida albicans and Candida glabrata. In addition to their antimicrobial activity, the anti-inflammatory potential for selected compounds was evaluated in silico and in vitro against human cyclooxygenase-2 (COX-2). The results showed that compounds 18, 21, 23, and 26 display in vitro inhibitory activity against COX-2, whereas docking calculations suggest a similar binding mode as compared to rofecoxib, the crystallographic reference.

Magnolia dealbata seeds extract exert cytotoxic and chemopreventive effects on MDA-MB231 breast cancer cells.

CONTEXT: Cancer prevention remains a high priority for the scientific world. Magnolia dealbata Zucc (Magnoliaceae), a Mexican endemic species, is used for the empirical treatment of cancer. OBJECTIVE: To evaluate the cytotoxic and cancer chemopreventive effects of an ethanol extract of Magnolia dealbata seeds (MDE). MATERIALS AND METHODS: The cytotoxic effect of MDE, at concentrations ranging from 1 to 200 µg/ml, on human cancer cells and human nontumorigenic cells was evaluated using the MTT assay for 48 h. The apoptotic activities of MDE 25 µg/ml on MDA-MB231 breast cancer cells were evaluated using the TUNEL assay and the detection of caspase 3 using immunofluorescence analysis for 48 h, each. The chemopreventive effect was evaluated by administrating different doses of MDE, between 1 and 50 mg/kg, injected intraperitoneally daily into athymic mice which were implanted with MDA-MB231 cells during 28 days. The growth and weight of tumors were measured. RESULTS: MDE showed cytotoxic effects on MDA-MB231 cells (IC50 = 25 µg/ml) and exerted pro-apoptotic activities as determined by DNA fragmentation in MDA-MB231 cells. MDE 25 µg/ml also induces the activation of caspase 3 in MDA-MB231 cells. These results suggest that Magnolia dealbata may be an optimal source of the bioactive compounds: honokiol (HK) and magnolol (MG). MDE 50 mg/kg i.p. exerted chemopreventive effects by inhibiting the growth of MDA-MB231 tumor by 75% in athymic mice, compared to the control group. CONCLUSIONS: MDE exerts cytotoxic, apoptotic and chemopreventive activities on MDA-MB231 human cancer cells.

Protector Role of Cx30.2 in Pancreatic ß-Cell against Glucotoxicity-Induced Apoptosis.

Glucotoxicity may exert its deleterious effects on pancreatic ß-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. ß-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in ß cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of ß cells (RIN-m5F). METHODS: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. RESULTS: High glucose did not induce apoptosis in RIN-m5F ß cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. CONCLUSION: The data suggest that the upregulation of Cx30.2 protects ß cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.

Metabolites from the Andean paramo lichen Cladonia cf. didyma and their effect as photoprotective and antioxidant agents.

5α,8α-peroxyergosteryl divaricatinate (1) is isolated for the first time from acetone extract of the thallus of Cladonia cf. didyma together with condidymic (2), isodidymic (3) and barbatic (4) acids. Their UVB/UVA photoprotective and antioxidant activities were determined along with their cyto- and DNA-protecting actions against OH• damage. Compound 1 showed high UVA, cyto- (%cell viability ca. 82 at 15 µM vs 71 for OH•) and DNA (%DNA-integrity ca. 74 at 100 µM vs 22 for OH•) protecting properties. Compounds 2 and 3 exhibited high UVB (SPF ca. 40) and antioxidant activities (free radical scavenging potency EC50 = 0.6694 and 1.700 mol compound/mol DPPH•; ferric reducing power 0.392 and 0.546, at 500 ppm and inhibited lipid peroxidation); protecting cells (%cell viability ca. 76 at 15 µM) and DNA (%DNA-integrity ca. 57 at 100 µM). Compounds 1 to 3 fulfilled some physicochemical properties to act topically on the skin.

Synthesis and Cytotoxic Activity of Combretastatin A-4 and 2,3-Diphenyl-2H-indazole Hybrids.

Cancer is the second leading cause of death, after cardiovascular diseases. Different strategies have been developed to treat cancer; however, chemotherapy with cytotoxic agents is still the most widely used treatment approach. Nevertheless, drug resistance to available chemotherapeutic agents is still a serious problem, and the development of new active compounds remains a constant need. Taking advantage of the molecular hybridization approach, in the present work we designed, synthesized, and tested the cytotoxic activity of two hybrid compounds and seven derivatives based on the structure of combretastatin A-4 and 2,3-diphenyl-2H-indazole. Practical modifications of reported synthetic protocols for 2-pheny-2H-indazole and 2,3-dipheny-2H-indazole derivatives under microwave irradiation were implemented. The cytotoxicity assays showed that our designed hybrid compounds possess strong activity, especially compound 5, which resulted even better than the reference drug cisplatin against HeLa and SK-LU-1 cells (IC50 of 0.16 and 6.63 µM, respectively), and it had similar potency to the reference drug imatinib against K562 cells. Additionally, in silico and in vitro studies strongly suggest tubulin as the molecular target for hybrid compound 5.

Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular modeling and structure-activity relationship of 9-anilinothiazolo[5,4-b]quinoline derivatives.

Some novel 9-anilinothiazolo[5,4-b]quinoline derivatives were synthesized and their cytotoxic activities were examined. The inhibition of some of the most active compounds over human topoisomerase II (Topo II) activity was assessed with the kDNA decatenation assay. The novel compounds differ in the substituents attached to the anilino ring, a dialkylamino alkylamino group, a saturated heterocyclic moiety, a methylthio group at position 2 and a fluorine atom present or absent at 7-position. According to the data, compounds with a diethylaminopropylamino group and a chlorine atom at 4'-position of the anilino ring were the most cytotoxic. The molecular models of all compounds indicated a correlation between hydrophobicity and cytotoxic activity although the direction and magnitude of the dipole moment also had a significant influence on its cytotoxicity. The 2-dialkylaminoalkylamino substituent is flexible and is known to facilitate the crossing of cell membranes; thus, this last barrier may be a limiting step in the mechanisms mediating the cytotoxicity. On the other hand, the activity of 2-methylthio derivatives seems to rely more on the electronic effects brought about by the substitution of the aniline ring. The synthesis, cytotoxicity against cancer cell lines, in vitro inhibition of human topoisomerase II, molecular modeling and the preliminary analysis of structure-activity relationships are presented.

Synthesis, cytotoxic evaluation, and DNA binding of novel thiazolo[5,4-b]quinoline derivatives.

A series of novel alkylamino and 9-anilinothiazolo[5,4-b]quinolines were synthesized as potential antitumoral agents. The in vitro cytotoxicity of these compounds was evaluated on several cell lines. The inclusion of electron-withdrawn/acceptor hydrogen-bond groups at position 3' of the anilino ring and the presence of an alkylamino chain on the tricyclic framework (regardless of its position) seem to be structural features relevant to cytotoxic activity.

Orcinol derivative compound with antioxidant properties protects Langerhans islets against streptozotocin damage.

OBJECTIVE: To design and synthesize an orcinol derivative compound, 3-formyl-2,4-dihydroxy-5,6-dimethyl sodium benzoate (A4), as an antioxidant molecule and to test its effects on oxidative stress in an in-vitro model of apoptosis of pancreatic rat beta cells induced by streptozotocin (STZ). METHODS: Scavenger properties of A4 were assessed using its capacity to capture the DPPH radical in vitro. Antiapoptotic properties of A4 were analysed by electron microscopy and TUNEL assay in rat pancreatic islets in a streptozotocin model. KEY FINDINGS: The results show that A4 displays antioxidant activity in in-vitro assays and induced a significant reduction in STZ-induced beta cell apoptosis and low ultrastructural damage to cellular organelles in the rat pancreatic islets as evidenced by electronic microscopy, this effect could be attributed to its antioxidant activity in a similar manner than resveratrol. CONCLUSION: The overall results indicate that the new orcinol derivative molecule displays both antioxidant and antiapoptotic effects and protect pancreatic beta cells against STZ damage.

Use of medicinal plants by health professionals in Mexico.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of medicinal plants in Mexico has been documented since pre-Hispanic times. Nevertheless, the level of use of medicinal plants by health professionals in Mexico remains to be explored. AIM OF THE STUDY: To evaluate the use, acceptance and prescription of medicinal plants by health professionals in 9 of the states of Mexico. MATERIALS AND METHODS: Direct and indirect interviews, regarding the use and acceptance of medicinal plants, with health professionals (n=1614), including nurses, physicians, pharmacists, and odontologists from nine states in Mexico were performed from January 2015 to July 2016. The interviews were analyzed with the factor the informant consensus (FIC). RESULTS: The information obtained indicated that 46% of those interviewed feel patients should not use medicinal plants as an alternative therapy. Moreover, 54% of health professionals, and 49% of the physicians have used medicinal plants as an alternative therapy for several diseases. Twenty eight percent of health professionals, and 26% of the physicians, have recommended or prescribed medicinal plants to their patients, whereas 73% of health professionals were in agreement with receiving academic information regarding the use and prescription of medicinal plants. A total of 77 plant species used for medicinal purposes, belonging to 40 botanical families were reported by the interviewed. The results of the FIC showed that the categories of diseases of the digestive system (FIC=0.901) and diseases of the respiratory system (FIC=0.898) had the greatest agreement. CONCLUSIONS: This study shows that medicinal plants are used for primary health care in Mexico by health professionals.

Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3ß.

OBJECTIVE: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. DESIGN: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated glycogen synthase kinase 3 beta (pGSK3ß), throughout the menstrual cycle. RESULTS: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3ß was identical in all samples and cycle phases, whereas pAkt and pGSK3ß, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. CONCLUSION: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways.

Molecular mechanisms involved in the cytotoxicity induced by coumarins from Calophyllum brasiliense in K562 leukaemia cells.

OBJECTIVES: The aim of this study was to determine the cellular and molecular mechanisms of cell death induced by mammea A/BA and A/BB (3 : 1) on K562 cells. METHODS: These compounds were isolated from Calophyllum brasiliense and its cytotoxicity was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell death was evaluated by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytofluorescence of active caspase-3. Genotoxicity was tested using comet assay. Lastly, a chemoinformatic analysis was performed with Osiris-Molinspiration software. KEY FINDINGS: The mixture of mammea A/BA and A/BB (3 : 1) showed cytotoxic activity against K562 cells (IC50 = 43.5 µm). TUNEL positive cells and active caspase-3 were detected after treatment. Genotoxicity of mammea A/BA and A/BB on K562 was detected since first hour of treatment. Additionally, mammea A/BA and A/BB were found to be in compliance with Lipinski 'rule of 5' suggesting that they possess strong potential of druglikeness. CONCLUSIONS: The overall results confirm and extend the knowledge about coumarins as an important resource of antitumor drugs, and indicate that these compounds could be used in further preclinical studies against leukaemia.

Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells.

Pachyrhizus erosus (Fabaceae) is a herb commonly known as 'yam bean', which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 µM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

In vitro drug metabolism testing using blood-monocyte derivatives.

INTRODUCTION: Monocytes and their cell derivatives can participate in drug metabolism. These cells express different Phase-I or -II drug metabolizing enzymes and can be differentiated into neo-hepatocytes (NeoHep) and represent a promising alternative strategy to test drug metabolism. This is particularly useful as primary human hepatocytes (PHH), are difficult to obtain and maintain in culture. AREAS COVERED: The authors analyze the use of blood monocytes and their derivatives for the study of drug metabolism. They also compare them to the in vitro ability of cells from different sources including: PHH, immortalized hepatocytes, tumor cell lines and NeoHep. EXPERT OPINION: The use of monocytes, macrophages, dendritic or Kupffer cells, to test drug metabolism, has serious limitations because these cells express lower levels of cytochrome P450 enzymes than PHH. The best available option, to replace PHH, have been tumor cell lines such as HepaRG, as well as immortalized hepatocytes from adult or fetal sources. Monocyte-derived NeoHep cells are novel and easily accessible cells, which express many drug metabolizing enzymes at levels comparable to PHH. These cells allow drug evaluation under a diverse genetic background. While these cells are in the early stages of evaluation and do need to be examined more thoroughly, they constitute a promising new tool for in vitro drug testing.

The products of the reaction between 6-amine-1,3-dimethyl uracil and bis-chalcones induce cytotoxicity with massive vacuolation in HeLa cervical cancer cell line.

As a part of our research in the chemistry of chalcones we have prepared four pyrimidine monoadducts of bis-chalcones through the reaction with 6-amino-1,3-dimethyl uracil. These compounds displayed cytotoxicity with a massive vacuolation in different human cell lines in vitro. Compound 6 was the most cytotoxic inducer of vacuoles, this compound induced G1 phase cell cycle arrest, and their cytotoxicity went without morphological and biochemical evidence of apoptotic cell death in HeLa cells. In addition, our results showed that this vacuole formation does not require de novo protein synthesis and the content vacuolar is acidic. Compound 6 induce necrotic cell death with excessive vacuolation, similar to a process of autophagy. Spautin-1 an inhibitor of autophagy, decreased the transformation of microtubule-associated protein 1 light chain 3 (LC3B-I) to LC3B-II and the vacuolation induced by compound 6 in HeLa cells, both autophagy processes. These compounds could be of pivotal importance in the study of non-apoptotic cell death with vacuole formation and could be useful in research into new autophagy inhibitors agents.

Kaempferitrin induces apoptosis via intrinsic pathway in HeLa cells and exerts antitumor effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Justicia spicigera is used for the empirical treatment of cervical cancer in Mexico. Recently, we showed that Justicia spicigera extracts exerted cytotoxic and antitumoral effects and the major component of this extract was kaempferitrin (KM). MATERIALS AND METHODS: The cytotoxic and apoptotic effect of KM on human cancer cells and human nontumorigenic cells were evaluated using MTT and TUNEL assays, and Annexin V/Propidium iodide detection by flow cytometry. The effect of KM on cell cycle was analyzed by flow cytometry with propidium iodide. The apoptotic and cell cycle effects were also evaluated by western blot analysis. Also, different doses of KM were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 32 days. The growth and weight of tumors were measured. RESULTS: KM induces high cytotoxic effects in vitro and in vivo against HeLa cells. The general mechanisms by which KM induces cytotoxic effects include: cell cycle arrest in G1 phase and apoptosis via intrinsic pathway in a caspase dependent pathway. Also, KM exerts chemopreventive and antitumor effects. CONCLUSION: KM exerts cytotoxic and antitumor effects against HeLa cells.

Synergistic anticancer activity of Thiazolo[5,4-b]quinoline derivative D3CLP in combination with cisplatin in human cervical cancer cells.

BACKGROUND: D3CLP (9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinoline) is a potent cytotoxic thiazolo[5,4-b]quinoline synthetic derivative that induces apoptosis of leukemia cells, while it displays low toxicity towards non-tumoral cells. The aim of this study was to determine if D3CLP can enhance the cytotoxicity of other antineoplastic drugs. MATERIALS AND METHODS: Leukemia, breast and cervical cancer cell lines were exposed to D3CLP-alone or in combination with imatinib, tamoxifen or cisplatin, respectively. Cell viability after treatment was evaluated by the MTT assay, and cell death by the TUNEL assay. The effects of combined treatments were analyzed by combination index and isobolographic analysis. RESULTS: Antiproliferative activity results indicate that D3CLP in combination with antineoplastic drugs induced a synergistic effect, at 3:1 and 1:1 ratios for D3CLP plus imatinib in K-562 leukemia cells, and at a 3:1 ratio for D3CLP with cisplatin in HeLa cells, as determined by their combination index. Furthermore, isobolographic analysis demonstrated a significant synergism for a 3:1 combination ratio of D3CLP with cisplatin in HeLa cells. In addition, TUNEL assay suggests cell death by apoptosis of HeLa cells after treatment with D3CLP and its combination with cisplatin at a 3:1 ratio. CONCLUSION: Overall the results indicate that D3CLP, in combined preparation with antineoplastic drugs, is a good candidate for pre-clinical studies in the treatment of different carcinoma cell types.

The antitumoral effect of the American mistletoe Phoradendron serotinum (Raf.) M.C. Johnst. (Viscaceae) is associated with the release of immunity-related cytokines.

ETHNOPHARMACOLOGICAL RELEVANCE: Phoradendron serotinum is commonly used in Mexican traditional medicine for the empirical treatment of cancer. However, there are no studies regarding the antitumoral or immunomodulatory activities of Phoradendron serotinum. MATERIALS AND METHODS: The in vivo toxicity of ethanolic extracts of Phoradendron serotinum (PSE) was evaluated in mice according to the Lorke procedure. The in vitro immunomodulatory effects of PSE were evaluated estimating the effects of PSE on the pinocytosis, NO production and lysosomal enzyme activity in murine macrophages RAW 264.7. The effects of PSE on the proliferation of murine splenocytes and NK cell activity were also assayed. The cytotoxic effects on TC-1 (lung murine cancer cells) were evaluated using the MTT assay, whereas the apoptotic effect of PSE on TC-1 cells was evaluated using TUNEL assay. Also, different doses of PSE were injected intraperitoneally daily into C57BL/6 mice bearing tumors of TC-1 cells during 25 days. The growth and weight of tumors was measured. In addition, the levels of IL-2, IL-6, IL-12, IL-23 and IFN-γ in murine serum and supernatants of K562 cell-murine splenocyte cocultures were measured. RESULTS: PSE stimulated the proliferation, pinocytosis and lysosomal enzyme activity in murine macrophages with a similar potency than lypopolisaccharides 1 µg/ml. In addition, PSE stimulated the proliferation of murine splenocytes and induced the NK cell activity. PSE showed cytotoxic (IC(50)=1.9 µg/ml) and apoptotic effects against TC-1 cells. The LD(50) was 125 mg/kg by intraperitoneal route (i.p.) and 375 mg/kg by oral route. PSE administrated at 1, 5 and 10 mg/kg i.p. inhibited the tumor growth by 18%, 40% and 69%, respectively, in mice bearing TC-1 tumor. PSE increased the in vitro and in vivo release of IL-2, IL-6 and IFN-γ but lacked effect on IL-12 and IL-23 release. CONCLUSIONS: Phoradendron serotinum shows moderate toxic effects in vivo, exerts cytotoxic and apoptotic effects on TC-1 cells. Phoradendron serotinum also has antitumor effects in mice bearing TC-1 tumor and induces immunomodulatory activities in vivo. The results suggest that antitumoral effects of PSE are related with the production of immunity-related cytokines.