Anticancer Activity and Molecular Targets of Piper cernuum Substances in Oral Squamous Cell Carcinoma Models.

Oral squamous cell carcinoma (OSCC) is a worldwide public health problem, with high morbidity and mortality rates. The development of new drugs to treat OSCC is paramount. Piper plant species have shown many biological activities. In the present study, we show that dichloromethane partition of Piper cernuum (PCLd) is nontoxic in chronic treatment in mice, reduces the amount of atypia in tongues of chemically induced OSCC, and significantly increases animal survival. To identify the main active compounds, chromatographic purification of PCLd was performed, where fractions 09.07 and 14.05 were the most active and selective. These fractions promoted cell death by apoptosis characterized by phosphatidyl serine exposition, DNA fragmentation, and activation of effector caspase-3/7 and were nonhemolytic. LC-DAD-MS/MS analysis did not propose matching spectra for the 09.07 fraction, suggesting compounds not yet known. However, aporphine alkaloids were annotated in fraction 14.05, which are being described for the first time in P. cernuum and corroborate the observed cytotoxic activity. Putative molecular targets were determined for these alkaloids, in silico, where the androgen receptor (AR), CHK1, CK2, DYRK1A, EHMT2, LXRß, and VEGFR2 were the most relevant. The results obtained from P. cernuum fractions point to promising compounds as new preclinical anticancer candidates.

New substances of Equisetum hyemale L. extracts and their in vivo antitumoral effect against oral squamous cell carcinoma.

ETHNOBOTANICAL RELEVANCE: Equisetum hyemale is used in traditional medicine as an anti-inflammatory, antioxidant, diuretic and anticancer agent. Recent studies have observed antiproliferative activity of this species in some tumor cell lines. AIM OF THE STUDY: The aim of this study was to evaluate the antiproliferative activity of the ethanol extract of E. hyemale and its partitions in oral squamous carcinoma cell lines, the death pathways induced by the most active partition, the acute toxicity and therapeutic activity, and the identification of the main compounds. MATERIALS AND METHODS: The ethanol crude extract was prepared from the stems of E. hyemale and partitions were obtained from this extract with n-hexane, dichloromethane and ethyl acetate. Cytotoxicity assays were performed using MTT on human oral tumor lines SCC-9, SCC4 and SCC-25, and normal primary fibroblasts. The main pathways of programmed cell death were analyzed. Acute toxicity in mice was performed using the most active partition, ethyl acetate. Antitumor activity was accessed in xenotransplants grafts of SCC-9 cells in Balb/nude mice. Phytochemical analysis was performed using UHPLC-MS/MS and dereplication was done using Global Natural Product Social Molecular Networking (GNPS) analysis. RESULTS: Ethanol extract, n-hexane and ethyl acetate partitions showed dose-dependent activity and selectivity towards oral tumor cells, with the ethyl acetate being the most bioactive. This medium polarity partition was shown to induce tumor cell death through apoptosis due to the presence of activated caspase 3/7, DNA fragmentation, chromatin condensation and phosphatidylserine exposure. The ethyl acetate partition also produced low toxicity in mice, provoking mild hepatic changes, but without causing necrosis and significantly reduced tumors volume and weight in xenotransplants of SCC-9 cells. Phytochemical analysis allowed identification of kaempferol glycosides and cinnamic acid derivatives previously described for E. hyemale. In addition it was possible to identify 6 new non-glycolyzed flavonoids 5-Hydroxy-3',4',7,8-tetramethoxyflavone (14), 5,4'-Dihydroxy-7,8,3'-trimethoxyflavone (15), 5,7-Dihydroxy-3',4'-dimethoxyflavone (16), 3',4,5,7-Tretramethoxyflavone (17), 5-Hydroxy-3'4',7-trimethoxyflavone (18), and 5,4'-Dihydroxy-3'-7'-dimethoxyflavone (19); besides 5 compounds already determined to be cytotoxic in other species, Isoferulic acid (1), Ferulic acid (2), Atractylenolide III (6), Dihydroxy-3',4'-dimethoxyflavone (16), and 5-Hydroxy-3'4 ',7-trimethoxyflavone (18). CONCLUSION: The results indicate that the E. hyemale extract and partitions inhibited 3 different cell lines of OSCC in a highly selective nontoxic way by inducing apoptosis of the cells. We identified 6 new non-glycosylated flavonoids and 5 other substances in this species.

Anti-Leishmania activity of extracts from Piper cabralanum C.DC. (Piperaceae).

Species of Piperaceae are known by biological properties, including antiparasitic such as leishmanicidal, antimalarial and in the treatment of schistosomiasis. The aim of this work was to evaluate the antileishmania activity, cytotoxic effect, and macrophage activation patterns of the methanol (MeOH), hexane (HEX), dichloromethane (DCM) and ethyl acetate (EtOAc) extract fractions from the leaves of Piper cabralanum C.DC. The MeOH, HEX and DCM fractions inhibited Leishmanina amazonensis promastigote-like forms growth with a half maximal inhibitory concentration (IC50) of 144.54, 59.92, and 64.87 µg/mL, respectively. The EtOAc fraction did not show any relevant activity. The half maximal cytotoxic concentration (CC50) for macrophages were determined as 370.70, 83.99, 113.68 and 607 µg/mL for the MeOH, HEX and DCM fractions, respectively. The macrophage infectivity was concentration-dependent, especially for HEX and DCM. MeOH, HEX and DCM fractions showed activity against L. amazonensis with low cytotoxicity to murine macrophages and lowering infectivity by the parasite. Our results provide support for in vivo studies related to a potential application of P. cabralanum extract and fractions as a promising natural resource in the treatment of leishmaniasis.

Cytotoxic effect of pure compounds from Piper rivinoides Kunth against oral squamous cell carcinoma.

The oral squamous cell carcinoma (OSCC) is the eighth more common cancer in men. The development of new and more efficient drugs is needed. Plants of the genus Piper are popularly used in the treatment of many diseases. This study evaluated the antitumor effect of extract, fraction and isolated compounds from leaves of P. rivinoides in oral cancer. The isolated compounds (conocarpan, eupomatenoid-5 and eupomatenoid-6) were effective in inducing cell death in OSCC cell lines (SCC4, SCC9 and SCC25) compared to the standard chemotherapeutic agent carboplatin, and this effect was time-dependent. Conocarpan was more selective and stable than eupomatenoid-5 and eupomatenoid-6, resembling the stability of carboplatin. There was a significant presence of pyknotic nuclei and active caspase-3 expression under conocarpan treatment, suggesting cell death through apoptosis. In conclusion, conocarpan was the most effective compound against OSCC cells and might be considered for future cancer studies.

Encapsulation of Piper cabralanum (Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells.

This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n-hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia.