piplartine and Triple-Negative-Breast-Neoplasms

TNBC exhibits higher rate of chemoresistance, metastasis, and relapse among all subtypes of breast cancer. This malignant statein TNBC is due to self-renewing sub-population of cells called cancer stem cells (CSCs). They are major caveats in TNBC treatment and need to be obliterated. In this regard, we explored piperlongumine (PL) that has remarkable anti-cancerous property but poor pharmacokinetics limits its application. So, to enhance its biological activity we developed PLGA based nanoformulation for PL (PL-NPs) and examined anti-CSCs effects of PL and PL-NPs in mammospheres. Results indicated that PL-NPs have higher cellular uptake than PL in mammospheres. Further, we demonstrated that PL-NPs remarkably inhibit various characteristics of CSCs like expression of ALDH, self-renewability, chemoresistance, and EMT in mammopsheres. We next investigated the possible mechanism underlying these multi-modal effects, and found that inhibition of STAT3 might be the driving force. In order to confirm this, we used colivelin a potent synthetic peptide activator of STAT3 in combination with treatments and found that anti-CSCs effects of PL and PL-NPs were reversed. Taken together, our data indicates that PL-NPs show enhanced inhibition of CSCs through downregulation of STAT3 and provides insight into development of PL based nanomedicine for targeting CSCs in TNBC.

Topics: Cell Line, Tumor; Cell Proliferation; Dioxolanes; Humans; Nanoparticles; Neoplastic Stem Cells; Spheroids, Cellular; STAT3 Transcription Factor; Triple Negative Breast Neoplasms; Tumor Cells, Cultured

In this study, we determined the effect of low dose piperlongumine on the motility/invasive capacity and epithelial-to-mesenchymal transition (EMT) of MDA-MB-231 triple-negative breast cancer (TNBC) cells and the metastasis of 4T1 mouse mammary carcinoma cells. MTT assays measured the effect of piperlongumine on TNBC cell growth. Motility/invasiveness were determined by gap closure/transwell assays. Western blotting assessed ZEB1, Slug, and matrix metalloproteinase (MMP) 9 expression. Interleukin (IL) 6 was detected by ELISA. MMP2, E-cadherin, and miR-200c expression was determined by real-time quantitative polymerase chain reaction. Reactive oxygen species (ROS) were measured by flow cytometry. The orthotopic 4T1 mouse model of breast cancer was used to examine metastasis. Piperlongumine-treated MDA-MB-231 cells showed reduced motility/invasiveness, decreased MMP2 and MMP9 expression, increased miR-200c expression, reduced IL-6 synthesis, decreased expression of ZEB1 and Slug, increased E-cadherin expression, and epithelial-like morphology. Piperlongumine also inhibited transforming growth factor β-induced ZEB1 and Slug expression. ROS accumulated in piperlongumine-treated cells, while changes in metastasis-associated gene expression were ablated by exogenous glutathione. Metastasis of 4T1 cells to the lungs of BALB/c mice was dramatically reduced in piperlongumine-treated animals. These findings reveal a previously unknown capacity of low dose piperlongumine to interfere with TNBC metastasis via an oxidative stress-dependent mechanism.

Topics: Alkaloids; Animals; Carcinoma; Cell Line, Tumor; Cell Movement; Dioxolanes; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mice; Triple Negative Breast Neoplasms

Triple-negative breast cancer (TNBC) lacks major effective target molecules and chemotherapy remains the current main treatment. However, traditional chemotherapy drugs, such as doxorubicin (DOX), cause serious side effects and have a poor prognosis. Piperlongumine (PL), a natural alkaloid, has showed selective anticancer effects and is expected to become a new strategy against TNBC. In our research, cell viability, colony formation, flow cytometry, Western blot, and tumor xenograft model assays were established to evaluate the suppression effect of PL and DOX alone and in combination. Data showed that PL could effectively inhibit cell growth and induce apoptosis in two TNBC cell lines. We also demonstrated for the first time that the combination treatment of PL and DOX synergistically inhibited cell growth and induced apoptosis in TNBC cells. The suppression of STAT3 activation was indicated to be a mechanism of the anticancer effect. Moreover, the effectiveness of this combination was confirmed in a tumor xenograft model. These results revealed that inhibition of the JAK2-STAT3 pathway was a key anticancer mechanism when treated with PL alone or combined with DOX, suggesting that the combination of PL and chemotherapy drugs may be a potential strategy for the clinical treatment of TNBC.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dioxolanes; Disease Models, Animal; Doxorubicin; Drug Synergism; Female; Humans; Janus Kinase 2; Mice; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Surgical removal of the primary tumor is a common practice in breast cancer treatment. However, postsurgical metastasis poses an immense setback in cancer therapy. Considering that 90% of cancer-related deaths are due to metastasis, antimetastatic therapeutic strategies that can target disseminating tumor cells in the circulation before they can form secondary tumors hold preclinical and clinical potential for cancer patients. Our current work uses a liposomal formulation functionalized with the adhesion receptor E-selectin and the apoptosis-inducing ligand TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL) to reduce metastasis following tumor resection in an aggressive triple-negative breast cancer (TNBC) mouse model. We demonstrate that minimal administration of E-selectin-TRAIL liposomes can target metastasis in a TNBC model, with primary tumor resection to mimic clinical settings. Our study indicates that TRAIL liposomes, alone or in combination with existing clinically approved therapies, may neutralize distant metastasis of a broad range of tumor types systemically.

Topics: Animals; Apoptosis; Aspirin; Cell Line, Tumor; Cell Proliferation; Curcumin; Dioxolanes; E-Selectin; Female; Humans; Leukocytes; Liposomes; Mammary Glands, Animal; Mice; Neoplasm Metastasis; Neoplasms, Experimental; TNF-Related Apoptosis-Inducing Ligand; Triple Negative Breast Neoplasms

Malignant transformation results in increased levels of reactive oxygen species (ROS). Adaption to this toxic stress allows cancer cells to proliferate. Recently, piperlongumine (PL), a natural alkaloid, was identified to exhibit novel anticancer effects by targeting ROS signaling. PL induces apoptosis specifically in cancer cells by downregulating several anti-apoptotic proteins. Notably, the same anti-apoptotic proteins were previously found to reduce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in cancer cells. Therefore, we reasoned that PL would synergize with TRAIL to stimulate potent apoptosis in cancer cells. We demonstrate for the first time that PL and TRAIL exhibit a synergistic anti-cancer effect in cancer cell lines of various origins. PL resulted in the upregulation of TRAIL receptor DR5, which potentiated TRAIL-induced apoptosis in cancer cells. Furthermore, such upregulation was found to be dependent on ROS and the activation of JNK and p38 kinases. Treatment with combined PL and TRAIL demonstrated significant anti-proliferative effects in a triple-negative breast cancer MDA-MB-231 xenograft model. This work provides a novel therapeutic approach for inducing cancer cell death. Combination of PL and TRAIL may suggest a novel paradigm for treatment of primary and metastatic tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Death; Cell Line, Tumor; Dioxolanes; Disease Models, Animal; Drug Synergism; Female; Humans; Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Very low oral bioavailability due to extensive pre-systemic metabolism and P-gp efflux has constrained the oral metronomic chemotherapy of docetaxel (DTX). There is tremendous need of compounds facilitating oral delivery of DTX. The research was aimed to investigate the effect of piperlongumine (PPL) on human liver microsomal metabolism, Caco-2 permeability, and cytotoxicity of DTX in triple-negative breast cancer cell lines. Reduction in testosterone and DTX metabolism (twofold increase in half-life) by PPL was comparable to the standard CYP3A4 inhibitor, cyclosporine A. P-gp efflux ratio of DTX across caco-2 monolayer was reduced from 2.37 to 1.52 on co-incubation with PPL. The IC50 value of DTX was reduced three to five times and combination index values in all the cell lines were below 0.6. PPL at non-cytotoxic concentration showed significant enhancement of the antimigration effect of DTX. Expression of tumor markers such as survivin, bcl2, C-myc, and cyclin D1 were downregulated to a great extent with enhanced p53 expression when treated with combination instead of individual drug. Co-treatment with PPL led to 1.68-fold enhancement in DTX bioavailability in SD rats. PPL could be a potential candidate in overcoming the obstacles associated with oral DTX delivery with synergistic anticancer activity.

Topics: Administration, Oral; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Availability; Caco-2 Cells; Cell Line, Tumor; Cyclosporine; Cytochrome P-450 CYP3A Inhibitors; Dioxolanes; Docetaxel; Drug Synergism; Female; Half-Life; Humans; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Taxoids; Triple Negative Breast Neoplasms

The phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3 K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3 K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3 K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3 K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3 k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer.

Topics: Alkaloids; Apoptosis; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Dioxolanes; DNA Fragmentation; Female; Humans; Molecular Docking Simulation; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms

The current study investigated the effects of Piper longumine on radio-sensitization of human breast cancer MDA-MB-231 cells and underlying mechanisms.. Human breast cancer MDA-MB-231 cells were cultured in vitro and those in logarithmic growth phase were selected for experiments divided into four groups: control, X-ray exposed, Piper longumine, and Piper longumine combined with X-rays. Conogenic assays were performed to determine the radio-sensitizing effects. Cell survival curves were fitted by single-hit multi-target model and then the survival fraction (SF), average lethal dose (D0), quasi-threshold dose (Dq) and sensitive enhancement ratio (SER) were calculated. Cell apoptosis was analyzed by flow cytometry (FCM).Western blot assays were employed for expression of apoptosis-related proteins (Bc1-2 and Bax) after treatment with Piper longumine and/or X-ray radiation. The intracellular reactive oxygen species (ROS) level was detected by FCM with a DCFH-DA probe.. The cloning formation capacity was decreased in the group of piperlongumine plus radiation, which displayed the values of SF2, D0, Dq significantly lower than those of radiation alone group and the sensitive enhancement ratio (SER) of D0 was1.22 and 1.29, respectively. The cell apoptosis rate was increased by the combination treatment of Piper longumine and radiation. Piper longumine increased the radiation-induced intracellular levels of ROS. Compared with the control group and individual group, the combination group demonstrated significantly decreased expression of Bcl-2 with increased Bax.. Piper longumine at a non-cytotoxic concentration can enhance the radio-sensitivity of MDA- MB-231cells, which may be related to its regulation of apoptosis-related protein expression and the increase of intracellular ROS level, thus increasing radiation-induced apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Survival; Dioxolanes; Female; Humans; Piper; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Radiation-Sensitizing Agents; Reactive Oxygen Species; Triple Negative Breast Neoplasms; X-Ray Therapy