[6]-Gingerol-Derived Semi-Synthetic Compound SSi6 Inhibits Tumor Growth and Metastatic Dissemination in Triple-Negative Breast Cancer Xenograft Models.

Breast cancer metastasis is the most common cause of cancer death in women worldwide. Triple-negative breast cancers (TNBC) form a heterogeneous group of tumors that have higher relapse rates and poorer survival compared to other breast cancer subtypes. Thus, this work reports the antitumor and antimetastatic activities of a [6]-gingerol-derived semi-synthetic compound named SSi6 on MDA-MB-231 TNBC cells using xenograft models. SSi6 did not cause toxic effects in vivo as demonstrated by body weight and hematological and histological evaluations. From the orthotopic xenograft model, we demonstrated that SSi6 slows and inhibits the growth of the primary tumor, as well as prevents metastatic spontaneous progression from lymph nodes to the lungs. Moreover, a second xenograft model with resection of the primary tumor showed that SSi6 also blocks the progression of metastases from the lymph nodes to other visceral organs. Taken together, our results demonstrate that SSi6 is a promising compound to be investigated in other preclinical and clinical models to be applied as a complementary therapy for TNBC.

[10]-Gingerol improves doxorubicin anticancer activity and decreases its side effects in triple negative breast cancer models.

PURPOSE: Although doxorubicin is widely used to treat cancer, severe side effects limit its clinical use. Combination of standard chemotherapy with natural products can increase the efficacy and attenuate the side effects of current therapies. Here we studied the anticancer effects of a combined regimen comprising doxorubicin and [10]-gingerol against triple-negative breast cancer, which does not respond to hormonal or targeted therapies. METHODS: Cytotoxicity was evaluated by MTT assay, cell cycle progression and apoptosis were analyzed by flow cytometry and signaling pathways were analyzed by Western blotting in human and murine triple negative breast cancer cell systems. The anticancer/antimetastatic and toxic effects of the combined regimen was evaluated using syngeneic and xenograft orthotopic models. RESULTS: The combination of doxorubicin and [10]-gingerol significantly increased the number of apoptotic cells, compared to each compound alone. In 4T1Br4 cells, the combined regimen was the only condition able to increase the levels of active caspase 3 and γH2AX and to decrease the level of Cdk-6 cyclin. In vivo, doxorubicin (3 mg/Kg, D3) and [10]-gingerol (10 mg/Kg, G10) resulted in a significant reduction in the volume of primary tumors and a decrease in the number of circulating tumor cells (CTCs). Interestingly, only the combined regimen led to decreased tumor burdens to distant organs (i.e., metastasis) and reduced chemotherapy-induced weight loss and hepatotoxicity in tumor-bearing animals. Likewise, in a xenograft model, only the combined regimen was effective in significantly reducing the primary tumor volume and the prevalence of CTCs. CONCLUSIONS: Our data indicate that [10]-gingerol has potential to be used as a neoadjuvant or in combined therapy with doxorubicin, to improve its anticancer activity.

Cytotoxicity of ruthenium-N,N-disubstituted-N'-acylthioureas complexes.

Five new complexes with general formula [Ru(Ln)(PP)(bipy)]PF6, where Ln = N,N'-dimethyl-N-Acyl thiourea, and P-P: 1,2-bis(diphenylphosphino)ethane (dppe) or 1,4-bis(diphenylphosphino)butane (dppb)) were synthesized and characterized by elemental analysis, molar conductivity, cyclic voltammetry, IR, NMR (1H, 13C{1H} and 31P{1H}), and single crystal X-ray diffractometry. The cytotoxicity of compounds against lung and breast tumor cell lines was significant, where two complexes, [Ru(L3)(bipy)(dppe)]PF6 (3) and [Ru(L3)(bipy)(dppb)]PF6 (6), were selected to evaluate changes in morphology, inhibition of migration and cell death in the MDA-MB-231 lineage. The complexes caused alterations in the cell morphology and were able to inhibit cell migration at the concentrations evaluated, induce the cell cycle arrested in the Sub-G1 phase, and induced cell death by apoptosis. All the complexes presented interaction with HSA, and the interaction studies with DNA suggested weak interactions, probably by the minor groove.

Structure Elucidation and Absolute Configuration Determination of Nortriterpenoids from Picramnia glazioviana.

In this study, HPLC-PDA-HRMS-SPE-NMR data were used for initial analysis of the CH2Cl2 fraction of an EtOH extract of the leaves of Picramnia glazioviana. The HRMS, UV, and NMR data obtained from the HPLC-PDA-HRMS-SPE-NMR analysis were used to direct semipreparative HPLC isolation toward nortriterpenoids, which resulted in the isolation of 18 new and highly oxygenated nortriterpenoids (1-3, 5-10, 12-19, and 21), named picravianes C-T. Their structures were determined on the basis of analysis of UV, HRMS, and 2D NMR spectroscopic data, including determination of the relative configuration on the basis of coupling pattern analysis and nuclear Overhauser effect correlations. The absolute configurations of compounds 7, 9, 10, 14, 15, 17, 18, 19, and 21 were assigned using electronic circular dichroism data, and the cytotoxicity of compounds 6, 10, 14, 16, 17, 18, 19, and 21 was evaluated against MDA-MB-231 triple-negative breast cancer, SKBR-3 Her2-overexpressing breast cancer, and A549 lung cancer cells lines. The isolated compounds contain a hitherto undescribed modification of the terminal backbone and/or E-ring, and a possible biosynthetic pathway for their formation is proposed.

Selective Coordination Mode of Acylthiourea Ligands in Half-Sandwich Ru(II) Complexes and Their Cytotoxic Evaluation.

In this study, half-sandwich Ru(II) complexes containing acylthiourea ligands of the general type [Ru(η6-p-cymene)(PPh3)(S)Cl]PF6 (1m-6m) and [Ru(η6-p-cymene)(PPh3)(S-O)]PF6 (1b-6b) where S/S-O = N',N'-disubstituted acylthiourea were synthesized and characterized (via elemental analyses, IR spectroscopy, 1H NMR spectroscopy, 13C{1H} NMR spectroscopy, and X-ray diffractometry), and their cytotoxic activity was evaluated. The different coordination modes of the acylthiourea ligands, monodentately via S (1m-6m) and bidentately via S,O (1b-6b), to ruthenium were modulated from different synthetic routes. The cytotoxicity of the complexes was evaluated in five human cell lines (DU-145, A549, MDA-MB-231, MRC-5, and MCF-10A) by MTT assay. The IC50 values for prostate cancer cells (2.89-7.47 µM) indicated that the complexes inhibited cell growth, but that they were less cytotoxic than cisplatin (2.00 µM). Unlike for breast cancer cells (IC50 = 0.28-0.74 µM) and lung cancer cells (IC50 = 0.51-1.83 µM), the complexes were notably more active than the reference drug, and a remarkable selectivity index (SI 4.66-19.34) was observed for breast cancer cells. Based on both the activity and selectivity, complexes 5b and 6b, as well as their respective analogous complexes in the monodentate coordination 5m and 6m, were chosen for further investigation in the MDA-MB-231 cell line. These complexes not only induced morphology changes but also were able to inhibit colony formation and migration. In addition, the complexes promoted cell cycle arrest at the sub-G1 phase inducing apoptosis. Interaction studies by viscosity measurements, gel electrophoresis, and fluorescence spectroscopy indicated that the complexes interact with the DNA minor groove and exhibit an HSA binding affinity.

Evaluation of the cytotoxicity on breast cancer cell of extracts and compounds isolated from Hyptis pectinata (L.) poit.

Hyptis pectinata is a herb popularly used in Brazil for the treatment of inflammations, pain, bacterial infections and cancer. In the present study, inflorescences (MPIn), leaves (MPL), branches (MPB), root (MPR) extracts and three compounds isolated from MPIn were assayed against breast tumor cell lines. The structures of the three compounds (pectinolide J, hyptolide and pectinolide E) were determined by means of spectroscopic analysis. Pectinolide J was isolated for the first time. The MPIn, MPL and MPR exhibited specific antiproliferative activity on tumor cell lines when compared to normal cell lines with IC50 of 52.01 ± 0.64, 45.91 ± 0.02 µg/mL and 82.84 ± 0.03 µg/mL, respectively. Although the isolated substances did not present good antiproliferative activity, when the three were associated, a greater biological effect was observed, suggesting a synergistic effect. Hyptolide (5.6 ± 0.4 µg/mL) showed IC50 sufficiently low to be considered as a drug prototype.

SSi6 promotes cell death by apoptosis through cell cycle arrest and inhibits migration and invasion of MDA-MB-231 human breast cancer cells.

Triple-negative breast cancer subtype is the most aggressive type of breast cancer due to the lack of specific therapeutic targets, having limited treatment options, low survival prognosis and high recurrence rates. In this work, we describe the effects of a semisynthetic derivative of [6]-gingerol (6G) called SSi6, produced by the addition of a 2,4-dinitrophenylhydrazine reagent on several aspects of triple-negative breast cancer biology. Human breast cancer cell lines MDA-MB-231 and MCF-10A were used in the experiments. MTT assays were used to detect cell viability. Cell cycle and apoptosis assay were analyzed using flow cytometer Accuri C6 and analysis of proteins as retinoblastoma Rb and kinases Cdk4/6 were analyzed by western blotting. SSi6 induced cytotoxic effects on triple-negative breast cancer cells, with higher selectivity when compared to the non-tumor MCF-10A cells. In addition, SSi6 inhibited migration and invasion of triple-negative breast cancer cells and was able to arrest cell cycle at the G1-phase, mainly by decreasing Cdk4/6-Rb axis levels. Therefore, SSi6 provoked the induction of apoptosis in triple-negative breast cancer cells. SSi6 was more efficient in producing these effects, compared to the original 6G natural product. This study may contribute to a better understanding of the effects of natural and semisynthetic products on the in-vitro metastatic processes in the MDA-MB-231 triple-negative breast cancer cell line. Additional, it can be useful to understand the effects of chemical modifications on already effective natural compounds aiming at the improvement of their bioactive properties, such as in the increase of the cytotoxic selectivity against tumor cells, compared to non-tumor ones.

Picraviane A and B: Nortriterpenes with limonoid-like skeletons containing a heptanolide E-ring system from Picramnia glazioviana.

Two highly oxygenated nortriterpenes, picraviane A and B, were isolated from the ethanolic extract of Picramnia glazioviana Engl. The structures were determined by analysis of HRMS and 2D NMR spectroscopic data. Single-crystal X-ray diffraction data was also obtained for picraviane B. The absolute configuration of both compounds were assigned by comparison of experimental and calculated vibrational and electronic circular dichroism spectroscopy, respectively. Picraviane A showed a moderate cytotoxic activity against the triple negative MDA-MB-231 breast cancer cell line. These compounds represent an undescribed class of natural products with limonoid-like skeletons containing a heptanolide as the E-ring.

Transport of the Ruthenium Complex [Ru(GA)(dppe)2]PF6 into Triple-Negative Breast Cancer Cells Is Facilitated by Transferrin Receptors.

The triple-negative breast cancer subtype (TNBC) is highly aggressive and metastatic and corresponds to 15-20% of diagnosed cases. TNBC treatment is hampered, because these cells usually do not respond to hormonal therapy, and they develop resistance to chemotherapeutic drugs. On the other hand, the severe side effects of cisplatin represent an obstacle for its clinical use. Ruthenium (Ru)-based complexes have emerged as promising antitumor and antimetastatic substitutes for cisplatin. In this study, we demonstrated the effects of a Ru/biphosphine complex, containing gallic acid (GA) as a ligand, [Ru(GA)(dppe)2]PF6, hereafter called Ru(GA), on a TNBC cell line, and compared them to the effects in a nontumor breast cell line. Ru(GA) complex presented selective cytotoxicity against TNBC over nontumor cells, inhibited its migration and invasion, and induced apoptosis. These effects were associated with the increased amount of transferrin receptors (TfR) on tumor cells, compared to nontumor ones. Silencing of TfR decreased Ru(GA) effects on TNBC cells, demonstrating that these receptors were at least partially responsible for Ru(GA) delivery into tumor cells. The Ru(GA) compound must be further studied in different in vivo assays in order to investigate its antitumor properties and its toxicity in complex biological systems.

Autophagy-dependent apoptosis is triggered by a semi-synthetic [6]-gingerol analogue in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is very aggressive and lacks specific therapeutic targets, having limited treatment options and poor prognosis. [6]-gingerol is the most abundant and studied compound in ginger, presenting diverse biological properties such as antitumor activity against several types of cancer, including breast cancer. In this study, we show that the semi-synthetic analogue SSi6, generated after chemical modification of the [6]-gingerol molecule, using acetone-2,4-dinitrophenylhydrazone (2,4-DNPH) reagent, enhanced selective cytotoxic effects on MDA-MB-231 cells. Remarkably, unlike the original [6]-gingerol molecule, SSi6 enabled autophagy followed by caspase-independent apoptosis in tumor cells. We found a time-dependent association between SSi6-induced oxidative stress, autophagy and apoptosis. Initial SSi6-induced reactive oxygen species (ROS) accumulation (1h) led to autophagy activation (2-6h), which was followed by caspase-independent apoptosis (14h) in TNBC cells. Additionally, our data showed that SSi6 induction of ROS plays a key role in the promotion of autophagy and apoptosis. In order to investigate whether the observed cell death induction was dependent on preceding autophagy in MDA-MB-231 cells, we used siRNA to knock down LC3B prior to SSi6 treatment. Our data show that LC3B downregulation decreased the number of apoptotic cells after treatment with SSi6, indicating that autophagy is a key initial step on SSi6-induced caspase-independent apoptosis. Overall, the results of this study show that structural modifications of natural compounds can be an interesting strategy for developing antitumor drugs, with distinct mechanisms of actions, which could possibly be used against triple negative breast cancer cells that are resistant to canonical apoptosis-inducing drugs.

Half sandwich Ru(ii)-acylthiourea complexes: DNA/HSA-binding, anti-migration and cell death in a human breast tumor cell line.

Organometallic ruthenium complexes as potential anticancer agents have been explored due to their suitable properties, such as stability in the solid state and in solution, water solubility and low toxicity. In this study, eight metal complexes of this class were synthesized, characterized and their important biological activities against a human breast tumor cell line (MDA-MB-231) were studied. Complexes 1-8 were obtained in good yields and have been characterized by satisfactory elemental analyses, IR, 1D and 2D 1H and 13C{1H} NMR, UV-Vis spectroscopy, cyclic voltammetry, ESI-MS and X-ray diffractometry (1, 2, 3 and 6). All complexes exhibit growth inhibition on human breast and lung tumor cell lines, with IC50 values ranging from 6.0 to 45.0 µM in 48 h. Four compounds were selected to evaluate the changes in the morphology, clonogenic, migration, cell cycle arrest and cell death in MDA-MB-231 cells. The complexes are able to induce morphological changes and inhibit the size, number of colonies and cell migration, and induce cell cycle arrest in the sub-G1 phase and apoptosis cell death. The interaction of the complexes with DNA was determined by performing spectroscopic titration, a competitive assay with thiazole orange, circular dichroism, gel electrophoresis and interactions with guanosine or guanosine monophosphate by 1H NMR, indicating the non-covalent interaction. The HSA binding affinity measured by spectrophotometric titration, revealed the hydrophobic and spontaneous association with the human protein. Overall, the studies indicated that these metal complexes are potential agents against MDA-MB-231 cells, encouraging us to continue studies of these types of compounds.