Negative regulation of signal transducer and activator of transcription-3 signalling cascade by lupeol inhibits growth and induces apoptosis in hepatocellular carcinoma cells.

BACKGROUND: Constitutive activation of signal transducer and activator of transcription signalling 3 (STAT3) has been linked with survival, proliferation and angiogenesis in a wide variety of malignancies including hepatocellular carcinoma (HCC). METHODS: We evaluated the effect of lupeol on STAT3 signalling cascade and its regulated functional responses in HCC cells. RESULTS: Lupeol suppressed constitutive activation of STAT3 phosphorylation at tyrosine 705 residue effectively in a dose- and time-dependent manner. The phosphorylation of Janus-activated kinases (JAKs) 1 and 2 and Src was also suppressed by lupeol. Pervanadate treatment reversed the downregulation of phospho-STAT3 induced by lupeol, thereby indicating the involvement of a phosphatase. Indeed, we observed that treatment with lupeol increased the protein and mRNA levels of SHP-2, and silencing of SHP-2 abolished the inhibitory effects of lupeol on STAT3 activation. Treatment with lupeol also downregulated the expression of diverse STAT3-regulated genes and decreased the binding of STAT3 to VEGF promoter. Moreover, the proliferation of various HCC cells was significantly suppressed by lupeol, being associated with substantial induction of apoptosis. Depletion of SHP-2 reversed the observed antiproliferative and pro-apoptotic effects of lupeol. CONCLUSIONS: Lupeol exhibited its potential anticancer effects in HCC through the downregulation of STAT3-induced pro-survival signalling cascade.

Modulation of humoral immune responses and inhibition of proinflammatory cytokines and nitric oxide production by 10-methoxycanthin-6-one.

Plant-derived natural products such as alkaloids, flavonoids, terpenoids, and polysaccharides have received considerable attention in recent years due to their diverse pharmacological properties such as immunomodulatory, anti-inflammatory, cytotoxic, cancer chemopreventive effects, and so on. 10-Methoxycanthin-6-one, a ß-carboline alkaloid from the medicinal plant Aerva lanata, was assessed for immunomodulatory activity in Balb/c mice. Intraperitoneal administration of five doses of the compound at 0.5 mg/kg body weight was found to enhance the total WBC count (13,975.50 ± 324.27 cells/mm³ on the 12th day), bone marrow cellularity (23.08 ± 0.86 × 106 cells/femur), and number of α-esterase-positive cells (1283.16 ± 21.10 cells/4000 cells). Treatment with the compound along with the antigen, sheep red blood cells, produced an enhancement in the circulating antibody titer (1024 on Days 12 and 15) and the number of plaque-forming cells (PFC) in the spleen. In treated group, maximum number of PFC (264.83 PFC/106 spleen cells) was observed on the sixth day after antigen administration. At the same time, administration of 10-methoxycanthin-6-one could significantly reduce the elevated levels of proinflammatory cytokines and nitric oxide production by lipopolysaccharide (LPS)-stimulated macrophages. There was also a significant reduction in the mRNA levels of inducible nitric oxide synthase, cyclooxygenase 2, tumor necrosis factor alpha (TNF)-α, and interleukin (IL)-1ß and IL-6 in LPS-stimulated macrophages after treatment with 10-methoxycanthin-6-one.

Immunomodulatory and antitumor activity of Aerva lanata ethanolic extract.

Cancer is responsible for millions of deaths each year worldwide. Pharmacological intervention with plant-derived products alone or in combination to reverse, suppress, or prevent the cancer progression plays a key role in the fight against this terrible disease. Aerva lanata is an important medicinal plant widely used in traditional systems of medicine like ayurveda and siddha. Ethanolic extract of whole plant of A. lanata exhibited immunomodulatory and antitumor activity. Intraperitoneal administration of five doses of the extract (10 mg/kg body weight) was found to enhance the total WBC count (14,238 cells/mm(3)), bone marrow cellularity (22.33 × 10(6) cells/femur), and number of α-esterase-positive cells (1276 cells/4000 cells). Aerva treatment also showed enhanced proliferation of splenocytes, thymocytes, and bone marrow cells both in the presence and absence of specific mitogens in vitro and in vivo. The number of plaque-forming cells (PFC) in spleen (243.33 PFC/10(6) spleen cells) and circulating antibody titer were also increased (P < 0.001). The extract was 100% cytotoxic to Dalton's lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC) cells at a concentration of 500 µg/mL. It was also found to be cytotoxic toward L929 and HELA cells at higher concentrations, whereas the nontoxic concentrations produced a reduction in the rate of proliferation. Simultaneous administration of five doses of A. lanata extract could produce significant inhibition in DLA-induced solid tumor development in mice and increase the life span of mice-bearing EAC tumors by 53.47%.

Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice.

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

Augmentation of humoral and cell mediated immune responses by Thujone.

Thujone, a naturally occurring monoterpene, was found to enhance the total WBC count, bone marrow cellularity, number of α-esterase positive cells, number of plaque forming cells in spleen and circulating antibody titer in Balb/c mice (1mg/kg body weight, intraperitoneally for 5 days). Thujone treatment enhanced proliferation of splenocytes and thymocytes, both in the presence and absence of specific mitogens. Administration of Thujone was found to stimulate the cell-mediated immunological response in normal and tumor bearing Balb/c mice. A significant enhancement in natural killer (NK) cell mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement mediated cytotoxicity (ACC) in both normal as well as tumor-bearing animals was observed after the administration of Thujone. Production of cytokines such as IL-2 and IFN-γ was significantly enhanced by the administration of Thujone. The stimulatory effect of Thujone on cytotoxic T lymphocyte (CTL) generation was determined by Winn's neutralization assay using CTL sensitive EL4 thymoma cells. Thujone treatment showed a significant increase in CTL production in both the in vivo and in vitro models, as indicated by a significant increase in the life span of tumor bearing animals. All these results indicate that administration of Thujone could enhance the immune response of mice. There was a significant reduction in solid tumor development, mediated by the presence of alert immune responses during Thujone administration.

Effect of amentoflavone, a phenolic component from Biophytum sensitivum, on cell cycling and apoptosis of B16F-10 melanoma cells.

Flavanoids are polyphenolic compounds that are found in fruits and vegetables and have diverse, beneficial biochemical and antioxidant effects. The objective of this study was to assess the effect of amentoflavone, a biflavanoid isolated from Biophytum sensitivum, on cell cycling distribution and apoptosis in B16F-10 melanoma cells. Treatment of B16F-10 melanoma cells with amentoflavone (10 µg/mL) increased cells in the sub-G0/G1 phase accompanied by a decrease in G0/G1 phase cells in a time-dependent manner. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of amentoflavone-treated B16F-10 melanoma cells confirmed that the cells were undergoing apoptosis. Amentoflavone was found to significantly inhibit B16F-10 melanoma-induced solid tumor development in C57BL/6 mice. The increase in apoptotic cells in paraffin sections obtained from amentoflavone-treated animals indicates that the reduction may be mediated through induction of apoptosis. Murine cell cycle-regulating genes, such as p21 and p27, and apoptosis-regulating genes, such as Bax and caspase-9, were found to be upregulated, whereas cyclin D1 and Bid were downregulated in amentoflavone-treated cells. These results demonstrate that amentoflavone can induce apoptosis via inhibiting progression of cells from G0/G1 to S phase and regulating genes involved in cell cycle regulation and apoptotic intrinsic pathways.

Role of macrophages in tumour progression.

It is now becoming clear that the inflammatory cells that exist in the tumour microenvironment play an indispensable role in cancer progression. Tumour associated macrophages (TAMs) represent a prominent component of the mononuclear leukocyte population of solid tumours, which displays an ambivalent relationship with tumours. They originate in the circulation and are recruited to the tumour site by tumour-derived attractants such as chemokines and interact with the tumour cells and preferentially localize at the tumour-host tissue interface, in regions often associated with low oxygen tensions. The tumour microenvironment, including cytokines and hypoxia, regulates the localization and function of TAMs. Upon activated by cancer cells, the TAMs can release a vast diversity of growth factors, proteolytic enzymes, cytokines, and inflammatory mediators. Many of these factors are key agents in cancer metastasis. Substantial evidence suggests that TAMs can interact with cancer cells, modify the ECM, and promote cancer cell invasion and metastasis. Several natural products have shown ability to inhibit the production of proinflammatory cytokines and growth factors by TAMs. The presence of extensive TAM infiltration has been shown to correlate with cancer metastasis and poor prognosis in a variety of human carcinomas.