Andrographolide induces apoptosis in B16F-10 melanoma cells by inhibiting NF-κB-mediated bcl-2 activation and modulating p53-induced caspase-3 gene expression.

Cancer is a disorder characterized by uncontrolled proliferation and reduced apoptosis. Inducing apoptosis is an efficient method of treating cancers. In this study, we investigated the effect of andrographolide on the induction of apoptosis as well as its regulatory effect on the activation of transcription factors in B16F-10 melanoma cells. Treatment of B16F-10 cells with nontoxic concentration of andrographolide showed the presence of apoptotic bodies and induced DNA fragmentation in a dose-dependent manner. Cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays also confirmed the observation. The proapoptotic genes p53, Bax, caspase-9, and caspase-3 were found upregulated in andrographolide-treated cells, whereas the antiapoptotic gene bcl-2 was downregulated. This study also reveals that andrographolide treatment could alter the production and expression of proinflammatory cytokines and could inhibit the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor-κB (NF-κB), and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein in B16F-10 melanoma cells. These results suggest that andrographolide induces apoptosis via inhibiting NF-κB-induced bcl-2-mediated survival signaling and modulating p53-induced caspase-3-mediated proapoptotic signaling.

Modulation of immune response by Vernonia cinerea L. inhibits the proinflammatory cytokine profile, iNOS, and COX-2 expression in LPS-stimulated macrophages.

The effect of methanolic extract of Vernonia cinerea L. on the immune system was studied using BALB/c mice. Intraperitoneal (i.p.) administration of five doses of the extract (20 mg/kg body weight) was found to enhance the total white blood cell (WBC) count (13,700 ± 463 cells/mm(3)) on 6th day, bone marrow cellularity (27.9 ± 2.1 × 10(6) cells/femur) and number of α-esterase positive cells (1184 ± 56.29/4000 cells). Treatment with V. cinerea along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titre and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (304.16 ± 12.4) was obtained on the 6th day. It also enhanced the proliferation of splenocytes, thymocytes and bone marrow cells both in the presence and absence of specific mitogens in vitro and in vivo. Administration of V. cinerea significantly reduced the lipopolysaccharide (LPS) induced elevated levels of nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor-α, interleukin-1 (IL-1ß), and IL-6 in mice. Treatment of V. cinerea methanolic extract also showed an enhancement in the phagocytic activity of peritoneal macrophages. Moreover The extract downregulated the inducible NO synthase and cyclooxygenase-2 (COX-2) mRNA expression in LPS-stimulated macrophages. These results indicate the immunomodulatory activity of V. cinerea.

Effect of vernolide-A, a sesquiterpene lactone from Vernonia cinerea L., on cell-mediated immune response in B16F-10 metastatic melanoma-bearing mice.

One of the major reasons for the rapid progression of cancers is the ability of tumor cells to escape from the immune surveillance mechanism of the body. Modulation of immune responses is highly relevant in tumor cell destruction. Effect of vernolide-A on the cell-mediated immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of vernolide-A enhanced natural killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared with the metastatic tumor-bearing control. Administration of vernolide-A significantly enhanced the production of interleukin (IL)-2 and interferon-gamma (IFN-γ) in metastatic tumor-bearing animals. In addition, vernolide-A significantly down-regulated the serum levels of proinflammatory cytokines such as IL-1ß, IL-6, tumor necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) during metastasis. All these results demonstrate that vernolide-A could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.

Ameliorative action of Vernonia cinerea L. on cyclophosphamide-induced immunosuppression and oxidative stress in mice.

Cyclophosphamide (CTX) is a widely used antineoplastic drug, which could cause toxicity to normal cells due to its toxic metabolites. The use of CTX in treating cancer patients is limited due to its severe toxicity induced mainly by oxidative stress. The present study reports the protective role of Vernonia cinerea L. against the CTX-induced toxicity in Balb/c mice. Intraperitoneal administration of the extract significantly increased the total WBC Count, bone marrow cellularity, alpha-esterase positive cells, and weights of lymphoid organs in CTX-treated animals, when compared with CTX control mice. Administration of V. cinerea was found to reduce the enhanced level of alkaline phosphatase, glutamate pyruvate transaminase, lipid peroxidation, and also significantly increased the reduced glutathione level in CTX-treated animals. Histopathological analysis of small intestine also suggests that extract could reduce the CTX-induced intestinal damage. The level of proinflammatory cytokine TNF-alpha, which was elevated during CTX administration, was significantly reduced by the V. cinerea extract administration. The lowered levels of other cytokines like IFN-gamma, IL-2, GM-CSF, after CTX treatment were also found to be increased by extract administration. Administration of V. cinerea did not compromise the anti-neoplastic activity of CTX. Infact, there was a synergistic action of CTX and V. cinerea in reducing the solid tumors in mice. Methanolic extract of V. cinerea given intraperitoneally (i.p.) showed a significant chemoprotective activity without compromising the chemotherapeutic efficacy of CTX, indicating its possible use as an adjuvant during chemotherapy.

Antimetastatic potential of vernolide-A, a sesquiterpenoid from Vernonia cinerea L.

The inhibitory effect of vernolide-A (C(21)H(28)O(7)) on lung metastasis induced by B16F-10 melanoma cells was studied using C57BL/6 mice. Vernolide-A was administered in three different modalities such as simultaneously with tumor, prophylactic to tumor and after tumor development. Maximum inhibition in the metastasis was observed when vernolide-A was administered simultaneously with tumor. There was 89.39% inhibition of lung tumor nodule formation and 88.51% increase in the life span of metastatic tumor-bearing animals. Highly elevated levels of lung hydroxyproline, lung uronic acid, lung hexosamine, serum sialic acid, serum γ-glutamyl transpeptidase (GGT) and serum vascular endothelial growth factor (VEGF) in the metastatic control animals were found to be significantly lowered in the vernolide-A-treated animals. Histopathological analysis of lung tissues also correlated with these results. Vernolide-A administration downregulated the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, extracellular-signal-regulated kinase-1 (ERK-1), ERK-2 and VEGF in the lung tissue of B16F-10 melanoma challenged animals. In the in vitro system, vernolide-A showed a significant inhibition of invasion of B16F-10 melanoma cells across the collagen matrix. Vernolide-A treatment also inhibited the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. Vernolide-A could inhibit MMP-2 and MMP-9 protein expression in gelatin zymographic analysis of B16F-10 cells. (3)H-thymidine proliferation assay showed that vernolide-A could inhibit the proliferation of B16F-10 melanoma cells in vitro. These results indicate that vernolide-A could inhibit the metastatic progression of B16F-10 melanoma cells in mice.

Protective role of Vernonia cinerea L. against gamma radiation--induced immunosupression and oxidative stress in mice.

The radioprotective effect of Vernonia cinerea extract was studied in balb/c mice. Whole-body irradiation of γ-rays (6 Gy) given to animals reduced the white blood cell count, bone marrow cellularity and α-esterase positive cells in control animals, which were elevated by the administration of V. cinerea extract (20 mg/kg body weight [b.wt.], intraperitoneally [i.p.]). The elevated levels of serum enzymes alkaline phosphatase (ALP), glutamate pyruvate transferases (GPT) and lipid peroxidation (LPO) after irradiation were also reduced with V. cineria extract administration. V. cinerea treatment also significantly enhanced the animal's antioxidant status by enhancing the activities superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) level in irradiated animals. Histopathological analysis of liver and small intestine also suggests that V. cinerea could reduce the tissue damages induced by radiation. The level of pro-inflammatory cytokines such as interleukin 1ß (IL-1ß), tumour necrosis factor α (TNF-α) and C-reactive protein (CRP) elevated after irradiation, which were significantly reduced by V. cinerea extract administration. On the other hand, the extract stimulated the production of other cytokines such as granulocyte monocyte-colony stimulating factor (GM-CSF) and interferon-γ (IFN-γ) in animals exposed to radiation. Agarose gel electrophoresis of DNA isolated from bone marrow of control animals showed heavy DNA damage, but a reduced DNA damage was seen in animals treated with V. cinerea extract. Administration of V. cinerea did not compromise the anti-neoplastic efficiency of radiation. In fact, there was a synergistic action of radiation and V. cinerea in reducing the solid tumours in mice. Methanolic extract of V. cinerea given i.p. showed a significant radioprotective activity without compromising the radiotherapeutic efficacy of radiation, indicating its possible use as an adjuvant during radiotherapy.

Oleanolic acid induces apoptosis by modulating p53, Bax, Bcl-2 and caspase-3 gene expression and regulates the activation of transcription factors and cytokine profile in B16F.

The objective of this study was to assess the effect of OA, a triterpene on inducing apoptosis in B16F-10 melanoma cells. Treatment of B16F-10 cells with nontoxic concentration of OA showed the presence of apoptotic bodies and induced DNA fragmentation in a dose-depended manner. Cell cycle analysis and TUNEL assay also confirmed the observation. The apoptotic genes p53, BAX, caspase-9, and caspase-3 were found upregulated in oleanolic acid­treated cells, whereas the antiapoptotic gene Bcl-2 was downregulated. OA treatment also showed a downregulation of Cyclin D1 expression and upregulated p21 and p27 gene expression in B16F-10 melanoma cells. OA inhibited the activation and nuclear translocation of transcription factors such asNF-κB, c-fos, ATF-2, and CREB-1, and also downregulated the production and expression of TNF-α, IL-1ß,IL-6, and GM-CS. These results suggest that OA induces apoptosis through activating the p53-induced caspase mediated proapoptotic pathway and suppression of the NF-κB­nduced Bcl-2­ediated antiapoptotic pathway

Andrographolide inhibits human umbilical vein endothelial cell invasion and migration by regulating MMP-2 and MMP-9 during angiogenesis.

Angiogenesis, the formation of new blood vessels from preexisting blood vessels, is essential for tumor progression and metastasis. Studies using human umbilical vein endothelial cells clearly demonstrated that administration of andrographolide significantly retarded endothelial cell proliferation, migration, and invasion and tube formation. Gelatin zymographic analysis showed the inhibitory effect of andrographolide on the activation of matrix metalloproteinases MMP-2 and MMP-9. The study reveals that andrographolide treatment could inhibit the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor-κB, and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element­binding protein in B16F-10 melanoma cells. All these results demonstrate that andrographolide inhibits in vitro angiogenesis by inhibiting MMP-2 and MMP-9, and also by regulating the nuclear translocation of transcription factors.

Vernolide-A inhibits radiation-induced hypoxia-mediated tumor angiogenesis by regulating HIF-1α, MMP-2, MMP-9, and VEGF.

We investigated the effect of vernolide-A on the inhibition of radiation-induced tumor angiogenesis in C57BL/6 mice. Vernolide-A administration significantly reduced the tumor volume of radiation-exposed mice. Serum vascular endothelial growth factor (VEGF) levels were drastically elevated during tumor progression and irradiation and were significantly reduced by treatment with vernolide-A. Immunohistochemical analysis also revealed reduced vascular density after treatment with vernolide-A, and 3H-thymidine incorporation assay and soft agar assay showed that vernolide-A could inhibit the proliferation of B16F-10 melanoma cells in vitro along with radiation. Vernolide-A also caused a significant inhibition in the invasion of irradiated B16F-10 melanoma cells across the collagen matrix, and inhibited the radiation-induced gene expression of hypoxia-inducible transcription factor-1α (HIF-1α) and VEGF in B16F-10 cells and VEGF receptor (Flk-1) expression in human umbilical vein endothelial cells. Gelatin zymographic analysis showed that vernolide-A could also inhibit the radiation-induced activation of matrix metalloproteinases (MMPs). Our results indicate that vernolide-A inhibits radiation-induced tumor angiogenesis by regulating HIF-1α, MMP-2, MMP-9, and VEGF.

Cardiospermum halicacabum inhibits cyclophosphamide induced immunosupression and oxidative stress in mice and also regulates iNOS and COX-2 gene expression in LPS stimulated macrophages.

The effect of a methanolic extract of Cardiospermum halicacabum L was studied against cyclophosphamide (CTX)-induced toxicity in mice. Administration of CTX (25 mg/kg b.wt, i.p.) for 10 days produced significant myelosuppression as evidenced by a decreased WBC count and bone marrow cellularity. Co-treatment with Cardiospermum significantly increased the total WBC count, bone marrow cellularity and α-esterase positive cells, and the relative organ weights of spleen as well as thymus compared to the CTX alone treated group. Cardiospermum further reduced the enhanced levels of ALP, GPT, LPO, and proinflammatory cytokine TNF-α, and also significantly increased the glutathione (GSH) level in CTX treated animals. The lowered levels of other cytokines like IFN-γ, IL-2, GM-CSF, after CTX treatment were also found to be increased by extract administration. Histopathological analysis of small intestine also suggested reduction of CTX-induced intestinal damage. Moreover the extract down-regulated the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) mRNA expression in LPS stimulated macrophages. These studies indicate that C. halicacabum could reduce cyclophosphamide induced oxidative stress and immunosupression through enhancing the antioxidant status and immunomodulation by stem cell proliferation.

Diallyl disulfide induces caspase-dependent apoptosis via mitochondria-mediated intrinsic pathway in B16F-10 melanoma cells by up-regulating p53, caspase-3 and down-regulating pro-inflammatory cytokines and nuclear factor-κß-mediated Bcl-2 activation.

Diallyl disulfide (DADS) is a major organo-sulfur compound derived from garlic (Allium sativum), which inhibits the proliferation of various types of cancer cells. In this study we investigated the effect of DADS on the induction of apoptosis, as well as its regulatory effect on the activation of transcription factors in B16F-10 melanoma cells. Treatment of B16F-10 cells with nontoxic concentrations of DADS resulted in the presence of apoptotic bodies and induced DNA fragmentation in a dose-dependent manner. Cell-cycle analysis revealed that the occurrence of the sub-G1 peak was significantly elevated in DADS-treated cells. DADS treatment also down-reguated Bcl-2 expression and up-regulated p53, caspase-9, and caspase-3 expression in B16F-10 melanoma cells. The study also reveals that DADS inhibited the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor (NF)-B and other transcription factors, such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein, in B16F-10 melanoma cells The pro-inflammatory cytokine production and gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were down-regulated in DADS-treated cells compared with control B16F-10 metastatic melanoma cells. DADS induces caspase-dependent apoptosis through a mitochondria-mediated intrinsic pathway in B16F-10 melanoma cells by activating p53 and caspase-3 gene expression and suppressing pro-inflammatory cytokines and NF-B-mediated Bcl-2 activation.

Protective role of perillic acid against radiation-induced oxidative stress, cytokine profile, DNA damage, and intestinal toxicity in mice.

The radioprotective effect of perillic acid was studied using an in vivo mouse model. Whole-body exposure of Swiss albino mice to γ-rays (6 Gy) reduced the total white blood cell count to 1035 ± 378 cells/mm3 on the 9th day, which was significantly elevated to 2196 ± 382 cells/mm3 by the administration of perillic acid (50 µmoles/kg body weight, intraperitoneally) on the same day. The number of bone marrow cells and α-esterase positive cells in control animals after 11 days of irradiation was reduced to 12.5 ± 0.8 × 106 cells/femur and 674 ± 45.2/4000 cells, respectively. In perillic acid treated animals, bone marrow cellularity was increased to 14.8 ± 1.8 × 106 cells/femur and α-esterase positive cells were 941 ± 56.5 /4000 cells, similar to normal level (935 ± 51.4/4000 cells). Administration of perillic acid could reduce the radiation-induced elevated levels of alkaline phosphatase (ALP), glutathione-pyruvate transferase (GPT) and lipid peroxidation (LPO) in both serum and liver of irradiated animals. Perillic acid could significantly enhance the glutathione (GSH) content in liver and intestinal mucosa of irradiated animals. Histopathological analysis of small intestine also suggests that perillic acid could reduce the radiation-induced intestinal damage. The level of proinflammatory cytokines such as IL-1ß, TNF-α and CRP, which were elevated during irradiation, was significantly reduced by the Perillic acid administration. Perillic acid treatment could also stimulate the production of other cytokines such as GM-CSF and IFN-γ in animals exposed to whole-body gamma irradiation. Agarose gel electrophoresis of DNA isolated from bone marrow of mice exposed to gamma radiation showed heavy damage that was reduced by treatment with perillic acid.