Kaempferol and Biomodified Kaempferol from Sophora japonica Extract as Potential Sources of Anti-Cancer Polyphenolics against High Grade Glioma Cell Lines.

The enzymatic hydrolysis of the extract of Sophora japonica by two glycosyl hydrolases (hesperidinase and galactosidase) was performed in order to obtain kaempferol (KPF)-enriched extract with an enhanced anticancer activity. The current study examined the effectiveness of both Sophora japonica extracts (before (KPF-BBR) and after (KPF-ABR) bioconversion reactions) in reducing cell viability and inducing apoptosis in human high-degree gliomas in vitro. Cytotoxicity was determined using an MTT assay. The effects of both compounds on the proliferation of glioma cell lines were measured using trypan blue exclusion, flow cytometry for cell cycle, wound healing (WH), and neurosphere formation assays. Cellular apoptosis was detected by DNA fragmentation and phosphatidylserine exposure. qPCR and luciferase assays evaluated NF-kB pathway inhibition. The survival rate of NG-97 and U-251 cells significantly decreased in a time- and dose-dependent manner after the addition of KPF-BBR or KPF-ABR. Thus, a 50% reduction was observed in NG-97 cells at 800 µM (KPF-BBR) and 600 µM (KPF-ABR) after 72 h. Both compounds presented an IC50 of 1800 µM for U251 after 72 h. The above IC50 values were used in all of the following analyses. Neither of the KPF presented significant inhibitory effects on the non-tumoral cells (HDFa). However, after 24 h, both extracts (KPF-BBR and KPF-ABR) significantly inhibited the migration and proliferation of NG-97 and U-251 cells. In addition, MMP-9 was downregulated in glioma cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus KPF-BBR and TPA+KPF-ABR compared with the TPA-treated cells. Both KPF-BBR and KPF-ABR significantly inhibited the proliferation of glioma stem cells (neurospheres) after 24 h. DNA fragmentation assays demonstrated that the apoptotic ratio of KPF-ABR-treated cell lines was significantly higher than in the control groups, especially NG-97, which is not TMZ resistant. In fact, the flow cytometric analysis indicated that KPF-BBR and KPF-ABR induced significant apoptosis in both glioma cells. In addition, both KPF induced S and G2/M cell cycle arrest in the U251 cells. The qPCR and luciferase assays showed that both KPFs downregulated TRAF6, IRAK2, IL-1ß, and TNF-α, indicating an inhibitory effect on the NF-kB pathway. Our findings suggest that both KPF-BBR and KPF-ABR can confer anti-tumoral effects on human cell glioma cells by inhibiting proliferation and inducing apoptosis, which is related to the NF-κB-mediated pathway. The KPF-enriched extract (KPF-ABR) showed an increased inhibitory effect on the cell migration and invasion, characterizing it as the best antitumor candidate.

Efficacy of Combined Use of Everolimus and Second-Generation Pan-EGRF Inhibitors in KRAS Mutant Non-Small Cell Lung Cancer Cell Lines.

Background: EGFR mutations are present in approximately 15−50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors' activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI50 score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI50 < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines.

Pharmacological and Therapeutic Potential of Myristicin: A Literature Review.

Natural products have been used by humanity for many centuries to treat various illnesses and with the advancement of technology, it became possible to isolate the substances responsible for the beneficial effects of these products, as well as to understand their mechanisms. In this context, myristicin, a substance of natural origin, has shown several promising activities in a large number of in vitro and in vivo studies carried out. This molecule is found in plants such as nutmeg, parsley, carrots, peppers, and several species endemic to the Asian continent. The purpose of this review article is to discuss data published in the last 10 years at Pubmed, Lilacs and Scielo databases, reporting beneficial effects, toxicity and promising data of myristicin for its future use in medicine. From 94 articles found in the literature, 68 were included. Exclusion criteria took into account articles whose tested extracts did not have myristicin as one of the major compounds.

Antiproliferative Effect of Synadenium grantii Hook f. stems (Euphorbiaceae) and a Rare Phorbol Diterpene Ester.

Synadenium grantii is frequently used for the treatment of various diseases such as allergies, gastric disorders, and especially cancer. The aim of this study was to evaluate the possible antiproliferative potential of the methanol extract, fractions, and pure compounds from the stems of S grantii Phytochemical analysis was carried out by conventional chromatographic techniques, and the antiproliferative activity was analyzed using the sulforhodamine B assay and an MTT-based assay. Nonpolar fraction and its subfractions from the stems of S grantii exhibited promising cytostatic effect against several human tumor cell lines (glioma, breast, kidney, and lung), with total grown inhibition values ranging from 0.37 to 2.9 µg/mL. One of the active principles of this plant was identified as a rare phorbol diterpene ester, denoted as 3,4,12,13-tetraacetylphorbol-20-phenylacetate. This compound demonstrated antiproliferative activity against glioma, kidney, lung, and triple-negative breast cancer cell lines. These results demonstrate that S grantii stems produce active principles with relevant antiproliferative potential.

Biflavonoids: Preliminary Reports on Their Role in Prostate and Breast Cancer Therapy.

Dimeric flavonoids, also called biflavonoids, are bioactive compounds that exhibit various activities described in the literature, including antibacterial, antifungal, antiviral, anti-inflammatory, analgesic, antioxidant, vasorelaxant, and anticancer properties. This work focuses on the anticancer action of naturally occurring dimeric flavonoids against prostate and breast cancer, as well as on the mechanisms of action involved in their activity and presents the most current information on this subject in the literature. In the present review, we summarize the latest findings on the antiproliferative activity of 33 dimeric flavonoid-based compounds selected from recently published studies. The tests conducted were in silico and in vitro and demonstrated the cytotoxic activity potential of biflavonoids against prostate and breast tumor cells. Biflavonoids were capable of interfering with the migration and replication of cancer cells and their mechanism of action is related to cell death pathways, especially apoptosis, necrosis, and ferroptosis. These compounds decreased mitochondrial membrane potential and significantly increased intracellular levels of reactive oxygen species (ROS). Additionally, they significantly upregulated the expression of p21, Bax, and cleaved caspase-3, while downregulating Bcl-2 and caspase-3 levels, indicating their cell death mechanism of action is through the Bcl-2/Bax/cleaved caspase-3 pathway and cell cycle arrest. The biflavonoids here related have shown promising anticancer activity and are considered potential drug candidates for prostate and breast cancer treatment.

Healing Potential of Propolis in Skin Wounds Evidenced by Clinical Studies.

Propolis has been used since ancient times for the treatment of skin diseases and, currently, its pharmacological potential for healing and repairing various types of wounds is widely cited in the literature. The healing properties of propolis are mainly attributed to its composition which is rich in phenolic compounds, and propolis has aroused the interest of the pharmaceutical industry as a low-cost product as compared with other treatments and medications; however, most of the published data refer to its effects in vitro and in vivo and, so far, few clinical studies have been carried out proving its therapeutic efficacy. In this article, we aimed to review clinical trail data published in Portuguese, Spanish, and English, in Scielo, PubMed, Google Scholar, Medline, and Lilacs between 1990 and 2021 on the clinical use of propolis for skin ulcers. The potential of propolis as an alternative healing treatment for skin wounds such as diabetic, venous, and surgical wounds, as well as wounds caused by burns, etc., is mainly due to its evidenced properties such as antimicrobial, anti-inflammatory, analgesic, and angiogenesis promoter effects. However, there is a need to standardize the type of administration and the concentration of propolis for each type of wound. Furthermore, further clinical studies are essential to add information about propolis safety and for obtaining the best possible therapeutic benefits from its use.

Effects of Myristicin in Association with Chemotherapies on the Reversal of the Multidrug Resistance (MDR) Mechanism in Cancer.

A range of drugs used in cancer treatment comes from natural sources. However, chemotherapy has been facing a major challenge related to multidrug resistance (MDR), a mechanism that results in a decrease in the intracellular concentration of chemotherapeutic agents, resulting in reduced treatment efficacy. The protein most frequently related to this effect is P-glycoprotein (P-gp), which is responsible for promoting drug efflux into the extracellular environment. Myristicin is a natural compound isolated from nutmeg and has antiproliferative activity, which has been reported in the literature. The present study aimed to evaluate the effect of the association between myristicin and chemotherapeutic agents on the NCI/ADR-RES ovarian tumor lineage that presents a phenotype of multidrug resistance by overexpression of P-gp. It was observed that myristicin showed no cytotoxic activity for this cell line, since its IC50 was >1 mM. When myristicin was associated with the chemotherapeutic agents cisplatin and docetaxel, it potentiated their cytotoxic effects, a result evidenced by the decrease in their IC50 of 32.88% and 75.46%, respectively. Studies conducted in silico indicated that myristicin is able to bind and block the main protein responsible for MDR, P-glycoprotein. In addition, the molecule fits five of the pharmacokinetic parameters established by Lipinski, indicating good membrane permeability and bioavailability. Our hypothesis is that, by blocking the extrusion of chemotherapeutic agents, it allows these agents to freely enter cells and perform their functions, stopping the cell cycle. Considering the great impasse in the chemotherapeutic treatment of cancer that is the MDR acquired by tumor cells, investigating effective targets to circumvent this resistance remains a major challenge that needs to be addressed. Therefore, this study encourages further investigation of myristicin as a potential reverser of MDR.

In vitro and in vivo anticancer activity of extracts, fractions, and eupomatenoid-5 obtained from Piper regnellii leaves.

Despite numerous studies with the Piper genus, there are no previous results reporting in vitro or in vivo Piper regnellii (Miq.) C. DC. var. regnellii anticancer activity. The aim of this study was to investigate P. regnellii in vitro and in vivo anticancer activity and further identify its active compounds. In vitro antiproliferative activity was evaluated in 8 human cancer cell lines: melanoma (UACC-62), breast (MCF7), kidney (786-0), lung (NCI-H460), prostate (PC-3), ovary (OVCAR-3), colon (HT29), and leukemia (K-562). Total growth inhibition (TGI) values were chosen to measure antiproliferative activity. Among the cell lines evaluated, eupomatenoid-5 demonstrated better in vitro antiproliferative activity towards prostate, ovary, kidney, and breast cancer cell lines. In vivo studies were carried out with Ehrlich solid tumor on Balb/C mice treated with 100, 300, and 1000 mg/kg of P. regnellii leaves dichloromethane crude extract (DCE), with 30 and 100 mg/kg of the active fraction (FRB), and with 30 mg/kg of eupomatenoid-5. The i. p. administration of DCE, FRB, and eupomatenoid-5 significantly inhibited tumor progression in comparison to control mice (saline). Therefore, this study showed that neolignans of Piper regnellii have promising anticancer activity. Further studies will be undertaken to determine the mechanism of action and toxicity of these compounds.

Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells.

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60µM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

Different cell death responses induced by eupomatenoid-5 in MCF-7 and 786-0 tumor cell lines.

Natural products remain an important source of new drugs, including anticancer drugs. Recently, our group reported the anticancer activity of eupomatenoid-5 (eup-5), a neolignan isolated from Piper regnellii (Miq.) C. DC. var. regnellii leaves. In vitro studies demonstrated that MCF-7 (breast) and 786-0 (kidney) were among the cancer cell lines most sensitive to eup-5 treatment. The current results demonstrate that mitochondrial membrane depolarization and generation of reactive oxygen species are implicated in eup-5-mediated cytotoxic effects on these cancer cells lines. In MCF-7 cells, eup-5 led to phosphatidylserine externalization and caspase activation, whereas the same did not occur in 786-0 cells. Scanning electron microscopy revealed a reduction of microvilli density, as well as cell morphology alterations. Moreover, treated MCF-7 cells exhibited well-characterized apoptosis alterations, while treated 786-0 cells exhibited characteristics of programmed necroptosis process. These findings support the possibility that different mechanisms may be targeted by eup-5 in cell death response.

Design and synthesis of N-acylated aza-goniothalamin derivatives and evaluation of their in vitro and in vivo antitumor activity.

Herein we describe the synthesis of a focused library of compounds based on the structure of goniothalamin (1) and the evaluation of the potential antitumor activity of the compounds. N-Acylation of aza-goniothalamin (2) restored the in vitro antiproliferative activity of this family of compounds. 1-(E)-But-2-enoyl-6-styryl-5,6-dihydropyridin-2(1H)-one (18) displayed enhanced antiproliferative activity. Both goniothalamin (1) and derivative 18 led to reactive oxygen species generation in PC-3 cells, which was probably a signal for caspase-dependent apoptosis. Treatment with derivative 18 promoted Annexin V/7-aminoactinomycin D double staining, which indicated apoptosis, and also led to G2 /M cell-cycle arrest. In vivo studies in Ehrlich ascitic and solid tumor models confirmed the antitumor activity of goniothalamin (1), without signs of toxicity. However, derivative 18 exhibited an unexpectedly lower in vivo antitumor activity, despite the treatments being administered at the same site of inoculation. Contrary to its in vitro profile, aza-goniothalamin (2) inhibited Ehrlich tumor growth, both on the ascitic and solid forms. Our findings highlight the importance of in vivo studies in the search for new candidates for cancer treatment.

Antitumor activity and toxicity of volatile oil from the leaves of Annona leptopetala

Abstract Annona leptopetala (R.E.Fr.) H. Rainer, Annonaceae, is used in folk medicine like antitumor and anti-inflammatory. The aim of this study was to determine chemical composition, toxicity and antitumor activity of A. leptopetala leaves volatile oil. Fresh leaves were hydrodistilled and then the volatile oil chemical composition was assessed by gas chromatography and mass spectrometry. Toxicity was assessed using haemolysis, micronucleus and acute toxicity protocols. Antitumor effects were determined in vitro and in vivo, using sulforhodamine B assay and sarcoma 180 murine tumor model, respectively. Spathulenol was the major component identified (12.56%). The volatile oil showed in vitro antitumor activity mainly in leukemia cell line (K-562), with Total growth inhibit (TGI) (concentration producing TGI) of 0.64 µg/ml. In other hand, the volatile oil <250 µg/ml did not inhibit HaCat non-tumor cell line growth. The concentration that produced 50% haemolysis was 372.8 µg/ml. The 50% lethal dose in mice was approximately 447.2 mg/kg intraperitoneally. Sarcoma 180 tumor growth inhibition rates were 59.29% and 58.77% at 100 and 150 mg/kg intraperitoneally, respectively. The volatile oil presented moderate gastrointestinal toxicity and no genotoxicity was observed at 350 mg/kg. Thus, the volatile oil shows antitumor activity with moderate toxicity.