Epoxyazadiradione exhibit anti‑cancer activities by modulating lncRNAs expression in pancreatic cancer

Background: Azadirachta indica (neem), a medicinal plant under Meliaceae family, is found in the Indian subcontinent. One of the limonoids, epoxyazadiradione (EPA), is a phytochemical isolated from the seeds of this tree. This is widely used in traditional medicine to treat a variety of human ailments. Although EPA has shown promise against some cancer types, its efficacy against pancreatic cancer and the underlying mechanism remains elusive. Aim: We examined the anti‑cancer activity of EPA against pancreatic cancer cells. We also examined the underlying mechanism. Methods: Pancreatic cancer cell lines (PANC-1 and MiaPaCa-2) were used during the study. We performed MTT assay, clonogenic colony formation assay for cytotoxicity. The western blotting was performed to examine the expression pattern of various apoptotic proteins. Real-time PCR was performed to detect quantitative lncRNAs expression. Results and Discussion: After treatment with EPA, the viability and proliferation of pancreatic cancer cells was decreased in a dose- and time-dependent manner. EPA suppressed the expression of apoptotic proteins involved in survival, proliferation, migration and invasion. EPA also suppressed the expression of MMP-9 in a concentration-dependent manner in pancreatic cancer cells. In addition, the limonoid also modulated the expression of lncRNAs (MEG-3, GAS-5, H19 and MHRT). Conclusion: EPA exhibited strong anti-cancer activities against pancreatic cancer by modulating multiple cancer-related signalling molecules

Molecular basis for the pharmacological activities of piperlongumine against breast cancer: Role of glucose import, ROS, NF-κB and lncRNAs

Background: Piperlongumine (PL, piplartine) is an alkaloid derived from the Piper longum L. (long pepper) root. The activities PL against breast cancer and the underlying mechanism is not thoroughly investigated. Aim: We examined the anti-cancer activities of PL against breast cancer cells. The molecular basis for the pharmacological activities of this alkaloid was also examined. Methods: The breast cancer cell lines such as MCF-7, T-47D, MDA-MB-231, MDA-MB-468 and MDA-MB-453 were used during the study. We used MTT assay, clonogenic and soft agar colony formation assay for cytotoxicity. The cell cycle analysis, phosphatidylserine externalization assay, measurement of mitochondrial membrane potential, AO/PI and DAPI staining, and DNA laddering was used for apoptosis. The western blot analysis was performed to examine the expression pattern of tumorigenic proteins. Other parameters used were the intracellular detection of ROS, immunocytochemistry for NF-κB and GLUT-1 activation, wound healing assay for cell migration, and real-time PCR for lncRNA expression. We also evaluated if PL can enhance the efficacy of doxorubicin in swiss albino mice implanted with Ehrlich Ascites Carcinoma (EAC) cells and metabolic parameters were also examined in serum of mice. Results: PL inhibited proliferation and suppressed the long-term as well as soft agar colony formation of breast cancer cells in a dose dependent manner. PL induced ROS generation and accumulation of cells in sub-G1 phase, mitochondria mediated apoptosis in cancer cells as revealed by the presence of fragmented nuclei, PARP activation, loss of mitochondrial membrane potential, chromatin condensation, DNA laddering and suppression in the expression of cell survival proteins. PL reduced glucose import and modifies the expression of glucose and lactate transporter in breast cancer cells. The amide alkaloid suppresses the TNF-α induced NF-κB activation and modulate the lncRNAs such as MEG-3, GAS-5 and H19 expression in breast cancer. In mice model, PL was found to synergize with doxorubicin by reducing the size, volume and weight of the tumor. With an increase in the concentration of PL, the serum cholesterol and triglyceride levels were decreased while there was increase in the serum level of glucose in EAC bearing mice. Conclusion: PL exhibit potential against breast cancer. Further, PL enhances the efficacy of doxorubicin in EAC mice model. The modulation of lncRNAs, NF-κB and glucose import may contribute to the activities of PL against breast cancer

Isodeoxyelephantopin, a Sesquiterpene Lactone Induces ROS Generation, Suppresses NF-κB Activation, Modulates LncRNA Expression and Exhibit Activities Against Breast Cancer

The sesquiterpene lactones, Isodeoxyelephantopin (IDET) and Deoxyelephantopin (DET) are known to exhibit activities against some cancer types. The activities of these lactones against breast cancer and the molecular bases is not known. We examined the efficacy of lactones in breast cancer preclinical model. Although both lactones exhibited drug like properties, IDET was relatively effective in comparison to DET. IDET suppressed the proliferation of both invasive and non-invasive breast cancer cell lines. IDET also suppressed the colony formation and migration of breast cancer cells. The assays for Acridine Orange (AO)/Propidium Iodide (PI) staining, cell cycle distribution, phosphatidylserine externalization and DNA laddering suggested the apoptosis inducing potential of IDET. The treatment with IDET also induced an accumulation of cells in the sub-G1 and G2/M phases. The exposure of breast cancer cells to the lactone was associated with a depolarization in mitochondrial membrane potential, and cleavage of caspase and PARP. The lactone induced reactive oxygen species (ROS) generation in breast cancer cells. Further, the use of N-acetyl cysteine (NAC) suppressed IDET induced ROS generation and apoptosis. The NF-κB-p65 nuclear translocation induced by okadaic acid (OA) was suppressed by the sesquiterpene. IDET also suppressed the expression of NF-κB regulated tumorigenic proteins, and induced the expression of proapoptotic gene (Bax) in cancer cells. While the expression of oncogenic lncRNAs was suppressed, the tumor suppressor lncRNAs were induced by the sesquiterpene. Collectively, the modulation of multiple cell signaling molecules by IDET may contribute to its activities in breast cancer cells

Targeting IκappaB kinases for cancer therapy

The inhibitory kappa B kinases (IKKs) and IKK related kinases are crucial regulators of the pro-inflammatory transcription factor, nuclear factor kappa B (NF-κB). The dysregulation in the activities of these kinases has been reported in several cancer types. These kinases are known to regulate survival, proliferation, invasion, angiogenesis, and metastasis of cancer cells. Thus, IKK and IKK related kinases have emerged as an attractive target for the development of cancer therapeutics. Several IKK inhibitors have been developed, few of which have advanced to the clinic. These inhibitors target IKK either directly or indirectly by modulating the activities of other signaling molecules. Some inhibitors suppress IKK activity by disrupting the protein-protein interaction in the IKK complex. The inhibition of IKK has also been shown to enhance the efficacy of conventional chemotherapeutic agents. Because IKK and NF-κB are the key components of innate immunity, suppressing IKK is associated with the risk of immune suppression. Furthermore, IKK inhibitors may hit other signaling molecules and thus may produce off-target effects. Recent studies suggest that multiple cytoplasmic and nuclear proteins distinct from NF-κB and inhibitory κB are also substrates of IKK. In this review, we discuss the utility of IKK inhibitors for cancer therapy. The limitations associated with the intervention of IKK are also discussed

Piperlongumine, a piper alkaloid, enhances the efficacy of doxorubicin in breast cancer: involvement of glucose import, ROS, NF-κB and lncRNAs

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

<i>Curcuma raktakanda</i> Induces Apoptosis and Suppresses Migration in Cancer Cells: Role of Reactive Oxygen Species

Although over 100 species of Curcuma are reported, only Curcuma longa is extensively studied. Curcuma raktakanda, a poorly studied species, is most commonly distributed in the Kerala state of India. For the first time, we examined the efficacy of different fractions (acetone, hexane, and ethyl acetate) of C. raktakanda against glioma, cervical, and breast cancer cell lines. As determined by mitochondrial reductase activity assay, the viability of cancer cells was decreased in a concentration-dependent manner by the three fractions. The half maximal inhibitory concentration (IC-50) values after the treatment of C-6 glioma cells for 48 h was found to be 32.97 &#181;g/mL (acetone extract), 40.63 &#181;g/mL (hexane extract), and 51.65 &#181;g/mL (ethyl acetate extract). Of the three fractions, the acetone fraction was more effective. The long-term colony formation of cancer cells was significantly suppressed by the acetone fraction. Analyses using DAPI (4&#8242;,6-diamidino-2-phenylindole) staining, AO/PI (acridine orange/propidium iodide) staining, DNA laddering, and sub-G1 population revealed that the acetone extract induced apoptosis in glioma cells. The extract induced reactive oxygen species generation and suppressed the expression of cell survival proteins. The migration of cancer cells was also suppressed by the acetone extract. The gas chromatography-mass spectrometry (GC-MS) analysis indicated that tetracontane, dotriacontane, hexatriacontane, pentacosane, hexacosane, and eicosane are the major components in the acetone extract. Collectively, the extract from C. raktakanda exhibited anti-carcinogenic activities in cancer cells. We are exploring whether the phytoconstituents, individually, or collectively contribute to the anti-cancer activities of C. raktakanda

Long non-coding RNAs are emerging targets of phytochemicals for cancer and other chronic diseases

10.1007/s00018-019-03053-0CELLULAR AND MOLECULAR LIFE SCIENCES76101947-196

Epoxyazadiradione exhibit activities in head and neck squamous cell carcinoma by targeting multiple pathways

The head and neck squamous cell carcinoma (HNSCC) constitute about 90% of all head and neck cancers. HNSCC falls in the top 10 cancers in men globally. Epoxyazadiradione (EPA) and Azadiradione (AZA) are the limonoids derived from the medicinal plant Azadirachta indica (popularly known as Neem). Whether or not the limonoids exhibit activities against HNSCC and the associated mechanism remains elusive. Herein, we demonstrate that EPA exhibits stronger activity in HNSCC in comparison to AZA. The limonoids obeyed the Lipinski\u27s rule of 5. EPA exhibited activities in a variety of HNSCC lines like suppression of the proliferation and the induction of apoptosis. The limonoid suppressed the level of proteins associated with anti-apoptosis (survivin, Bcl-2, Bcl-xL), proliferation (cyclin D1), and invasion (MMP-9). Further, the expression of proapoptotic Bax and caspase-9 cleavage was induced by the limonoid. Exposure of EPA induced reactive oxygen species (ROS) generation in the FaDu cells. N-acetyl-L-cysteine (ROS scavenger) abrogated the down-regulation of tumorigenic proteins caused by EPA exposure. EPA induced NOX-5 while suppressing the expression of programmed death-ligand 1 (PD-L1). Further, hydrogen peroxide induced NF-κB-p65 nuclear translocation and EPA inhibited the translocation. Finally, EPA modulated the expression of lncRNAs in HNSCC lines. Overall, these results have shown that EPA exhibit activities against HNSCC by targeting multiple cancer related signalling molecules. Currently, we are evaluating the efficacy of this molecule in mice models