piplartine and Breast-Neoplasms

Piperlongumine (PL, piplartine) is an alkaloid derived from the Piper longum L. (long pepper) roots. Originally discovered in 1961, the biological activities of this molecule against some cancer types was reported during the last decade. Whether PL can synergize with doxorubicin and the underlying mechanism in breast cancer remains elusive. Herein, we report the activities of PL in numerous breast cancer cell lines. PL reduced the migration and colony formation by cancer cells. An enhancement in the sub-G1 population, reduction in the mitochondrial membrane potential, chromatin condensation, DNA laddering and suppression in the cell survival proteins was observed by the alkaloid. Further, PL induced ROS generation in breast cancer cells. While TNF-α induced p65 nuclear translocation, PL suppressed the translocation in cancer cells. The expression of lncRNAs such as MEG3, GAS5 and H19 were also modulated by the alkaloid. The molecular docking studies revealed that PL can interact with both p65 and p50 subunits. PL reduced the glucose import and altered the pH of the medium towards the alkaline side. PL also suppressed the expression of glucose and lactate transporter in breast cancer cells. In tumor bearing mouse model, PL was found to synergize with doxorubicin and reduced the size, volume and weight of the tumor. Overall, the effects of doxorubicin in cancer cells are enhanced by PL. The modulation of glucose import, NF-κB activation and lncRNAs expression may have contributory role for the activities of PL in breast cancer.

Topics: Alkaloids; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dioxolanes; Doxorubicin; Female; Glucose; Humans; Mice; Molecular Docking Simulation; NF-kappa B; Piper; Reactive Oxygen Species; RNA, Long Noncoding

Advances in the early diagnosis and treatment have led to increases in breast cancer survivorship. Survivors report cognitive impairment symptoms such as loss of concentration and learning and memory deficits which significantly reduce the patient's quality of life. Additional therapies are needed to prevent these side effects and, the precise mechanisms of action responsible are not fully elucidated. However, increasing evidence points toward the use of neuroprotective compounds with antioxidants and anti-inflammatory properties as tools for conserving learning and memory. Here, we examine the ability of piperlongumine (PL), an alkaloid known to have anti-inflammatory and antioxidant effects, to play a neuroprotective role in 16-week-old female C57BL/6J mice treated with a common breast cancer regimen of doxorubicin, cyclophosphamide, and docetaxel (TAC). During social memory testing, TAC-treated mice exhibited impairment, while TAC/PL co-treated mice did not exhibit measurable social memory deficits. Proteomics analysis showed ERK1/2 signaling is involved in TAC and TAC/PL co-treatment. Reduced Nrf2 mRNA expression was also observed. mRNA levels of Gria2 were increased in TAC treated mice and reduced in TAC/PL co-treated mice. In this study, PL protects against social memory impairment when co-administered with TAC via multifactorial mechanisms involving oxidative stress and synaptic plasticity.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Breast Neoplasms; Chemotherapy-Related Cognitive Impairment; Cognitive Dysfunction; Dioxolanes; Female; Inflammation; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Quality of Life; RNA, Messenger; Signal Transduction

Nanoemulsions modified with chitosan (NE-Q) or hyaluronic acid (NE-HA), developed for intraductal administration of piplartine (piperlongumine) and local breast cancer treatment, were evaluated for cytotoxic effects in vitro in 2D and 3D breast cancer models and in vivo in a chemically induced carcinogenesis model. Droplet size was lower than 100 nm, and zeta potential varied from +17.9 to -25.5 mV for NE-Q and NE-HA, respectively. Piplartine nanoencapsulation reduced its IC

Topics: Breast Neoplasms; Chitosan; Female; Humans; Hyaluronic Acid; Nanoparticles; Piperidones

Breast cancer has become a worldwide threat, and chemotherapy remains a routine treatment. Patients are forced to receive continuous chemotherapy and suffer from severe side effects and poor prognosis. Natural alkaloids, such as piperine (PP) and piperlongumine (PL), are expected to become a new strategy against breast cancer due to their reliable anticancer potential. In the present study, cell viability, flow cytometry, and Western blot assays were performed to evaluate the suppression effect of PP and PL, alone or in combination. Data showed that PP and PL synergistically inhibited breast cancer cells proliferation at lower doses, while only weak killing effect was observed in normal breast cells, indicating a good selectivity. Furthermore, apoptosis and STAT3 signaling pathway-associated protein levels were analyzed. We demonstrated that PP and PL in combination inhibit STAT3 phosphorylation and regulate downstream molecules to induce apoptosis in breast cancer cells. Taken together, these results revealed that inactivation of STAT3 was a novel mechanism with treatment of PP and PL, suggesting that combination application of natural alkaloids may be a potential strategy for prevention and therapy of breast cancer.

Topics: Alkaloids; Apoptosis; Benzodioxoles; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dioxolanes; Female; Humans; MCF-7 Cells; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Signal Transduction; STAT3 Transcription Factor

Piperlongumine (PL), a natural alkaloid compound isolated from long pepper (

Topics: A549 Cells; Antineoplastic Agents; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Death; Dioxolanes; Gene Expression; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-fos; Reactive Oxygen Species; RNA, Small Interfering

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerized when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, showing potent effects in human breast carcinoma MCF-7 cells whilst being relatively non-toxic to non-tumorigenic MCF-10a cells. These compounds will be further developed as potential clinical candidates for the treatment of breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Survival; Dioxolanes; Female; Humans; MCF-7 Cells; Microtubules; Models, Molecular; Piperidones; Reactive Oxygen Species; Structure-Activity Relationship; Tubulin; Tubulin Modulators

HER family receptors are frequently deregulated in breast cancer and the deregulation of these receptors is associated with poor prognosis. Thus, these receptors are considered therapeutic targets. In the present study, we found that piperlongumine (PL) downregulates the expression of HER family receptors HER1, HER2, and HER3 in breast cancer cells. Downregulation of these receptors by PL is mediated through the generation of reactive oxygen species (ROS), as N-acetyl-cysteine blocks it. Interestingly, the HER2-overexpressing cell lines BT474 and SkBr3 are somewhat more sensitive to PL than the low HER2-expressing cell line MCF7. In addition, the overexpression of HER2 increases the sensitivity of MCF7 cells to PL. Collectively, our data indicate the therapeutic potential of PL in the treatment of breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Dioxolanes; Dose-Response Relationship, Drug; Down-Regulation; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Reactive Oxygen Species

Small molecules that can restore biological function to the p53 mutants found in human cancers have been highly sought to increase the anticancer efficacy. In efforts to generate hybrid anticancer drugs that can impact two or more targets simultaneously, we designed and developed piperlongumine (PL) derivatives with an aryl group inserted at the C-7 position. This insertion bestowed a combretastatin A4 (CA4, an established microtubule disruptor) like structure while retaining the piperlongumine configuration. The new compounds exhibited potent antiproliferative activities against eight cancer cell lines, in particular, were more cytotoxic against the SKBR-3 breast cancer cells which harbor a R175H mutation in p53 suppressor. KSS-9, a representative aryl PL chosen for further studies induced abundant ROS generation and protein glutathionylation. KSS-9 strongly disrupted the tubulin polymerization in vitro, destabilized the microtubules in cells and induced a potent G2/M cell cycle block. More interestingly, KSS-9 showed the ability to reactivate the p53 mutation and restore biological activity to the R175H mutant protein present in SKBR3 cells. Several procedures, including immunocytochemistry using conformation-specific antibodies for p53, immunoprecipitation combined with western blotting, electrophoretic shift mobility shift assays showed a reciprocal loss of mutant protein and generation of wild-type like protein. p53 reactivation was accompanied by the induction of the target genes, MDM2, p21cip1 and PUMA. Mechanistically, the redox-perturbation in cancer cells by the hybrid drug appears to underlie the p53 reactivation process. This anticancer drug approach merits further development.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Chemistry Techniques, Synthetic; Dioxolanes; Drug Design; Female; Genes, Tumor Suppressor; Glutathione; Humans; Microtubules; Mutation; Reactive Oxygen Species; Stilbenes; Tumor Suppressor Protein p53

Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been used to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. PL inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that PL directly inhibited binding of Stat3 to its phosphotyrosyl peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as Janus kinases, Src kinase family members or receptor tyrosine kinases. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient-derived xenografts. PL's antitumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 has a role.

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Proliferation; Dioxolanes; Drug Repositioning; Female; Gene Expression; Humans; MCF-7 Cells; Mice; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Oxidative Stress; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spheroids, Cellular; STAT3 Transcription Factor; Surface Plasmon Resonance; Transplantation, Heterologous; Tumor Cells, Cultured

Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic α,β-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

Topics: Apoptosis; Breast Neoplasms; Dioxolanes; Enzyme Induction; Female; Heme Oxygenase-1; Humans; MCF-7 Cells; NF-E2-Related Factor 2; Reactive Oxygen Species; Signal Transduction

The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling.. We examined Akt/mTOR signalling in cancer cells of various origins including prostate, kidney and breast after PL treatment. Furthermore, cell viability after concomitant treatment with PL and the autophagy inhibitor, Chloroquine (CQ) was assessed. We then examined the efficacy of in vivo combination treatment using a mouse xenograft tumour model.. We demonstrate for the first time that PL effectively inhibits phosphorylation of Akt target proteins in all tested cells. Furthermore, the downregulation of Akt downstream signalling resulted in decrease of mTORC1 activity and autophagy stimulation. Using the autophagy inhibitor, CQ, the level of PL-induced cellular death was significantly increased. Moreover, concomitant treatment with PL and CQ demonstrated notable antitumour effect in a xenograft mouse model.. Our data provide novel therapeutic opportunities to mediate cancer cellular death using PL. As such, PL may afford a novel paradigm for both prevention and treatment of malignancy.

Topics: Animals; Apoptosis; Autophagy; Breast Neoplasms; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chloroquine; Dioxolanes; Female; HEK293 Cells; Humans; Kidney Neoplasms; Male; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Neoplasm Transplantation; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Piperlongumine (PL) has been shown to selectively induce apoptotic cell death in cancer cells via reactive oxygen species (ROS) accumulation. In this study, we characterized a molecular mechanism for PL-induced cell death.. Cell viability and cell death were assessed by MTT assay and Annexin V-FITC/PI staining, respectively. ROS generation was measured using the H2DCFDA. Small interfering RNA (siRNA) was used for suppressing gene expression. The mRNA and protein expression were analyzed by RT-PCR and Western blot analysis, respectively.. We found that PL promotes C/EBP homologous protein (CHOP) induction, which leads to the up-regulation of its targets Bim and DR5. Pretreatment with the ROS scavenger N-acetyl-cysteine abolishes the PL-induced up-regulation of CHOP and its target genes, suggesting an essential role for ROS in PL-induced CHOP activation. The down-regulation of CHOP or Bim with siRNA efficiently attenuates PL-induced cell death, suggesting a critical role for CHOP in this cell death. Furthermore, PL potentiates TRAIL-induced cytotoxicity in breast cancer cells by upregulating DR5, as DR5 knockdown abolished the sensitizing effect of PL on TRAIL responses.. Overall, our data suggest a new mechanism for the PL-induced cell death in which ROS mediates CHOP activation, and combination treatment with PL and TRAIL could be a potential strategy for breast cancer therapy.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Breast Neoplasms; Cell Line, Tumor; Dioxolanes; Female; Humans; Membrane Proteins; Proto-Oncogene Proteins; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Comet Assay; Dioxolanes; DNA Damage; Genotype; Mice; Neoplasm Metastasis; Oxidative Stress; Reactive Oxygen Species; Small Molecule Libraries; Xenograft Model Antitumor Assays