Massimo Tacchini

Hemidesmus indicus induces apoptosis as well as differentiation in a human promyelocytic leukemic cell line

ETHNOPHARMACOLOGICAL RELEVANCE The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever, and diarrhea. Poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. The aim of this study was to investigate the cytodifferentiative, cytostatic and cytotoxic potential of a decoction of Hemidesmus indicuss roots (0.31-3 mg/mL) on a human promyelocytic leukemia cell line (HL-60). MATERIALS AND METHODS The decoction of Hemidesmus indicus was characterized by HPLC to quantify its main phytomarkers. Induction of apoptosis, cell-cycle analysis, levels of specific membrane differentiation markers were evaluated by flow cytometry. The analysis of cell differentiation by nitroblue tetrazolium (NBT) reducing activity, adherence to the plastic substrate, α-napthyl acetate esterase activity and morphological analysis was performed through light microscopy (LM) and transmission electron microscopy (TEM). RESULTS Starting from the concentration of 0.31 mg/ml, Hemidesmus indicus induced cytotoxicity and altered cell-cycle progression, through a block in the G0/G1 phase. The decoction caused differentiation of HL-60 cells as shown by NBT reducing activity, adherence to the plastic substrate, α-naphtyl acetate esterase activity, and increasing expression of CD14 and CD15. The morphological analysis by LM and TEM clearly showed the presence of granulocytes and macrophages after Hemidesmus indicus treatment. CONCLUSIONS The cytodifferentiating, cytotoxic and cytostatic activities of Hemidesmus indicus offers a scientific basis for its use in traditional medicine. Its potent antileukemic activity provides a pre-clinical evidence for its traditional use in anticancer pharmacology. Further experiments are worthwhile to determine the in vivo anticancer potential of this plant decoction and its components.

Research Progress in the Modification of Quercetin Leading to Anticancer Agents

The flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone) is widely distributed in plants, foods, and beverages. This polyphenol compound exhibits varied biological actions such as antioxidant, radical-scavenging, anti-inflammatory, antibacterial, antiviral, gastroprotective, immune-modulator, and finds also application in the treatment of obesity, cardiovascular diseases and diabetes. Besides, quercetin can prevent neurological disorders and exerts protection against mitochondrial damages. Various in vitro studies have assessed the anticancer effects of quercetin, although there are no conclusive data regarding its mode of action. However, low bioavailability, poor aqueous solubility as well as rapid body clearance, fast metabolism and enzymatic degradation hamper the use of quercetin as therapeutic agent, so intense research efforts have been focused on the modification of the quercetin scaffold to obtain analogs with potentially improved properties for clinical applications. This review gives an overview of the developments in the synthesis and anticancer-related activities of quercetin derivatives reported from 2012 to 2016.

Phytochemical profile and bioactivity of traditional ayurvedic decoctions and hydro-alcoholic macerations of Boerhaavia diffusa L. and Curculigo orchioides Gaertn.

Decoctions (DECs) and hydro-alcoholic extracts (HEs) prepared from roots of Boerhaavia diffusa L. (Nyctaginaceae) and Curculigo orchioides Gaertn. (Hypoxidaceae) were phytochemically characterised by HPLC-DAD and profiled for their antioxidant, antigenotoxic and cytotoxic activities. B. diffusa DEC was rich in ferulic acid and vanillin, while the HE also contained boeravinone B and eupalitin. Both C. orchioides HE and DEC displayed the main occurrence of orcinol-β-d-glucoside and curculigoside A. Antioxidant activity was assayed through spectrophotometric DPPH, ABTS and β-carotene bleaching test, and using (HP)TLC bioautographic strategies. For both crude drugs, HE was the best performing preparation. Properly modified SOS-Chromotest evidenced a 10% inhibition by phytocomplexes against 4-nitroquinoline-N-oxide, and a higher bioactivity for vanillin (36.60 ± 1.68%) and ferulic acid (35.09 ± 1.53%). C. orchioides HE was the preparation which showed higher cytotoxicity against drug-sensitive human T-lymphoblastoid cell line (CCRF-CEM) and multidrug-resistant leukaemia cell line (CEM/ADR5000), and eupalitin was the only pure compound to exhibit an IC50 value.

Withania somnifera Induces Cytotoxic and Cytostatic Effects on Human T Leukemia Cells

Cancer chemotherapy is characterized by an elevated intrinsic toxicity and the development of drug resistance. Thus, there is a compelling need for new intervention strategies with an improved therapeutic profile. Immunogenic cell death (ICD) represents an innovative anticancer strategy where dying cancer cells release damage-associated molecular patterns promoting tumor-specific immune responses. The roots of Withania somnifera (W. somnifera) are used in the Indian traditional medicine for their anti-inflammatory, immunomodulating, neuroprotective, and anticancer activities. The present study is designed to explore the antileukemic activity of the dimethyl sulfoxide extract obtained from the roots of W. somnifera (WE). We studied its cytostatic and cytotoxic activity, its ability to induce ICD, and its genotoxic potential on a human T-lymphoblastoid cell line by using different flow cytometric assays. Our results show that WE has a significant cytotoxic and cytostatic potential, and induces ICD. Its proapoptotic mechanism involves intracellular Ca2+ accumulation and the generation of reactive oxygen species. In our experimental conditions, the extract possesses a genotoxic potential. Since the use of Withania is suggested in different contexts including anti-infertility and osteoarthritis care, its genotoxicity should be carefully considered for an accurate assessment of its risk–benefit profile.

Multi-target activity of Hemidesmus indicus decoction against innovative HIV-1 drug targets and characterization of Lupeol mode of action

Despite the availability of several anti-retrovirals, there is still an urgent need for developing novel therapeutic strategies and finding new drugs against underexplored HIV-1 targets. Among them, there are the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) function and the cellular α-glucosidase, involved in the control mechanisms of N-linked glycoproteins formation in the endoplasmic reticulum. It is known that many natural compounds, such as pentacyclic triterpenes, are a promising class of HIV-1 inhibitors. Hence, here we tested the pentacyclic triterpene Lupeol, showing that it inhibits the HIV-1 RT-associated RNase H function. We then performed combination studies of Lupeol and the active site RNase H inhibitor RDS1759, and blind docking calculations, demonstrating that Lupeol binds to an HIV-1 RT allosteric pocket. On the bases of these results and searching for potential multitarget active drug supplement, we also investigated the anti-HIV-1 activity of Hemidesmus indicus, an Ayurveda medicinal plant containing Lupeol. Results supported the potential of this plant as a valuable multitarget active drug source. In fact, by virtue of its numerous active metabolites, H. indicus was able to inhibit not only the RT-associated RNase H function, but also the HIV-1 RT-associated RNA-dependent DNA polymerase activity and the cellular α-glucosidase.

Myrcia splendens (Sw.) DC. (syn. M. fallax (Rich.) DC.) (Myrtaceae) essential oil from amazonian Ecuador: A chemical characterization and bioactivity profile

In this study, we performed the chemical characterization of Myrcia splendens (Sw.) DC. (Myrtaceae) essential oil from Amazonian Ecuador and the assessment of its bioactivity in terms of cytotoxic, antibacterial, and antioxidant activity as starting point for possible applicative uses. M. splendens essential oil, obtained by hydro-distillation, was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID): the major components were found to be trans-nerolidol (67.81%) and α-bisabolol (17.51%). Furthermore, we assessed the cytotoxic activity against MCF-7 (breast), A549 (lung) human tumor cell lines, and HaCaT (human keratinocytes) non-tumor cell line through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test: promising results in terms of selectivity and efficacy against the MCF-7 cell line (IC50 of 5.59 ± 0.13 μg/mL at 48 h) were obtained, mainly due to α-bisabolol. Furthermore, antibacterial activity against Gram positive and negative bacteria were performed through High Performance Thin Layer Chromatography (HPTLC) bioautographic assay and microdilution method: trans-nerolidol and β-cedren-9-one were the main molecules responsible for the low antibacterial effects against human pathogens. Nevertheless, interesting values of Minimum Inhibitory Concentration (MIC) were noticeable against phytopathogen strains. Radical scavenging activity performed by HPTLC bioautographic and spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) approaches were negligible. In conclusion, the essential oil revealed a good potential for plant defense and anti-cancer applications.

Inhibition of Cancer Cell Proliferation and Antiradical Effects of Decoction, Hydroalcoholic Extract, and Principal Constituents of Hemidesmus indicus R. Br.

Indian Sarsaparilla (Hemidesmus indicus R. Br.) is widely used in Indian traditional medicine. In the present work, we explored the effects of decoction, traditional Ayurvedic preparation, and hydroalcoholic extract, a phytocomplex more traditionally studied and commercialized as food supplement in western medicine, from the roots as possible source of chemicals with new functional potential linked to their nutritional uses. The antiproliferative and antioxidant properties were assayed. To test antiproliferative affects, different cancer cell lines, growing both as monolayers (CaCo2, MCF‐7, A549, K562, MDA‐MB‐231, Jurkat, HepG2, and LoVo) and in suspension (K562 and Jurkat) were used. The decoction showed strong activity on HepG2 cells, while the hydroalcoholic extracts were active on HepG2, LoVo, MCF‐7, K562, and Jurkat cell lines. Weak inhibition of cancer cell proliferation was observed for the principal constituents of the preparations: 2‐hydroxy‐4‐methoxybenzaldehyde, 2‐hydroxy‐4‐methoxybenzoic acid, and 3‐hydroxy‐4‐methoxybenzaldehyde that were tested alone. The antiradical activity was tested with 2,2‐diphenyl‐1‐picrylhydrazyl and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid)diammonium salt tests and inhibition of nitric oxide production in lipopolysaccharide‐stimulated RAW 264.7 macrophages. Interesting result has also been obtained for hydroalcoholic extract regarding genoprotective potential (58.79% of inhibition at 37.5 µg/mL). Copyright

In Vitro Study of the Cytotoxic, Cytostatic, and Antigenotoxic Profile of Hemidesmus indicus (L.) R.Br. (Apocynaceae) Crude Drug Extract on T Lymphoblastic Cells

In traditional Indian medicine, the crude drug Hemidesmus indicus root—commonly known as Indian sarsaparilla—is used alone or in poly-herbal preparations for the treatment of a wide range of diseases. The present study focuses on the cancer chemopreventive and therapeutic potential of H. indicus extracts on an acute lymphoblastic leukemia cell line (CCRF-CEM). With this aim in mind, we subjected H. indicus roots to two subsequent extractions (hydro-alcoholic extraction and soxhlet extraction). As DNA damage is an important prerequisite for the induction of mutations/cancer by genotoxic carcinogens, cancer chemoprevention may be achieved by preventing genotoxicity. Through an integrated experimental approach, we explored the genoprotective potential of the soxhlet H. indicus extract against different mutagenic compounds and its cytotoxic, proapoptotic, and cytostatic properties. In our experimental conditions, H. indicus induced a cytotoxic effect involving the activation of both intrinsic and extrinsic apoptotic pathways and blocked the cell cycle in the S phase. Moreover, the antigenotoxicity results showed that the extract was able to mitigate DNA damage, an essential mechanism for its applicability as a chemopreventive agent, via either the modulation of extracellular and intracellular events involved in DNA damage. These data add to the growing body of evidence that H. indicus can represent a noteworthy strategy to target early and late stages of cancer.

Inhibitory effect of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. essential oils from Ecuador on West Nile virus infection

Abstract West Nile virus (WNV) is a mosquito-borne flavivirus responsible of neuroinvasive manifestations. Natural products are well-known for their biological activities and pharmaceutical application. In this study, the inhibitory effects of essential oils (EOs) of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. on WNV replication were investigated. WNV was incubated with EOs before adsorption on Vero cells, viral replication was carried out in the absence or presence of EO. Cells were exposed to EO before the adsorption of untreated-virus. GC-MS and GC-FID were used for chemical characterization of EOs. Cell protection from infection was observed for both EOs. P. aduncum EO was characterized by dillapiole as main compound (48.21%) and O. quixos EO by 1,8-cineole (39.15%). Further investigations, such as the study of molecular and cellular mechanisms of action and in vivo evaluation, should be performed on these essential oils to derive new potential drugs against WNV.

Hemidesmus indicus induces immunogenic death in human colorectal cancer cells

The ability of anticancer treatments to promote the activation of tumor-reactive adaptive immune responses is emerging as a critical requirement underlying their clinical effectiveness. We investigated the ability of Hemidesmus indicus, a promising anticancer botanical drug, to stimulate immunogenic cell death in a human colorectal cancer cell line (DLD1). Here we show that Hemidesmus treatment induces tumor cell cytotoxicity characterized by surface expression of calreticulin, increased HSP70 expression and release of ATP and HMGB1. Remarkably, the exposure to released ICD-inducer factors from Hemidesmus-treated DLD1 cells caused a modest induction of CD14-derived dendritic cells maturation, as demonstrated by the increased expression of CD83. Moreover, at sub-toxic concentrations, H.i. treatment of monocytes and dendritic cells induced their mild activation, suggesting its additional direct immunostimulatory activity. These data indicate that Hemidesmus indicus induces immunogenic cell death in human tumor cells and suggest its potential relevance in innovative cancer immunotherapy protocols.