Simultaneous determination of cucurbitacins B, D, and E in Cucurbita seed oils by HPLC-DAD.

Cucurbitacins are triterpene bioactive constituents of natural products, particularly in the Cucurbitaceae plant family. The presence of cucurbitacins in seeds of the Cucurbita genus (pumpkin) has been only little studied. In this work, the content of cucurbitacins B, D, and E in seed oils from three cucurbits (Cucurbita moschata Duch, Cucurbita pepo Linn, and Cucurbita maxima Linn) was studied. An analytical method based on HPLC-DAD for the detection and quantification of these three cucurbitacins in seed oils was developed and validated according to ICH guidelines. The method showed good linearity, accuracy, and precision for the simultaneous quantification of cucurbitacins B, D, and E using C.moschata seed oil as a reference. When applied to C.pepo and C.maxima seed oils, cucurbitacin B and D were quantified but to a lesser extent. This is the first report of a simple, repeatable, and reproducible analytical tool to identify cucurbitacins in oilseeds from Cucurbita spp.

Investigation of apoptotic effects of Cucurbitacin D, I, and E mediated by Bax/Bcl-xL, caspase-3/9, and oxidative stress modulators in HepG2 cell line.

Cucurbitacins, natural compounds highly abundant in the Cucurbitaceae plant family, are characterized by their anticancer, anti-inflammatory, and hepatoprotective properties. These compounds have potential as therapeutic agents in the treatment of liver cancer. This study investigated the association of cucurbitacin D, I, and E (CuD, CuI, and CuE) with the caspase cascade, Bcl-2 family, and oxidative stress modulators in the HepG2 cell line. We evaluated the antiproliferative effects of CuD, CuI, and CuE using the MTT assay. We analyzed Annexin V/PI double staining, cell cycle, mitochondrial membrane potential, and wound healing assays at different doses of the three compounds. To examine the modulation of the caspase cascade, we determined the protein and gene expression levels of Bax, Bcl-xL, caspase-3, and caspase-9. We evaluated the total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), glutathione (GSH), Total, and Native Thiol levels to measure cellular redox status. CuD, CuI, and CuE suppressed the proliferation of HepG2 cells in a dose-dependent manner. The cucurbitacins induced apoptosis by increasing caspase-3, caspase-9, and Bax activity, inhibiting Bcl-xL activation, causing loss of ΔΨm, and suppressing cell migration. Furthermore, cucurbitacins modulated oxidative stress by increasing TOS levels and decreasing SOD, GSH, TAS, and total and native Thiol levels. Our findings suggest that CuD, CuI, and CuE exert apoptotic effects on the hepatocellular carcinoma cell line by regulating Bax/Bcl-xL, caspase-3/9 signaling, and causing intracellular ROS increase in HepG2 cells.

Analgesic Effect of SH003 and Trichosanthes kirilowii Maximowicz in Paclitaxel-Induced Neuropathic Pain in Mice.

Pacliatxel is a taxol-based chemotherapeutic drug that is widely used to treat cancer. However, it can also induce peripheral neuropathy, which limits its use. Although several drugs are prescribed to attenuate neuropathies, no optimal treatment is available. Thus, in our study, we analyzed whether SH003 and its sub-components could alleviate paclitaxel-induced neuropathic pain. Multiple paclitaxel injections (cumulative dose 8 mg/kg, i.p.) induced cold and mechanical allodynia from day 10 to day 21 after the first injection in mice. Oral administration of SH003, an herbal mixture extract of Astragalus membranaceus, Angelica gigas, and Trichosantheskirilowii Maximowicz (Tk), dose-dependently attenuated both allodynia. However, when administered separately only Tk decreased both allodynia. The effect of Tk was shown to be mediated by the spinal noradrenergic system as intrathecal pretreatment with α1- and α2-adrenergic-receptor antagonists (prazosin and idazoxan), but not 5-HT1/2, and 5-HT3-receptor antagonists (methysergide and MDL-72222) blocked the effect of Tk. The spinal noradrenaline levels were also upregulated. Among the phytochemicals of Tk, cucurbitacin D was shown to play a major role, as 0.025 mg/kg (i.p.) of cucurbitacin D alleviated allodynia similar to 500 mg/kg of SH003. These results suggest that Tk should be considered when treating paclitaxel-induced neuropathic pain.

The involvement of the noradrenergic system in the antinociceptive effect of cucurbitacin D on mice with paclitaxel-induced neuropathic pain.

Paclitaxel (sold under the brand name Taxol) is a chemotherapeutic drug that is widely used to treat cancer. However, it can also induce peripheral neuropathy, which limits its use. Although several drugs are used to attenuate neuropathy, no optimal treatment is available to date. In this study, the effect of cucurbitacins B and D on paclitaxel-induced neuropathic pain was assessed. Multiple paclitaxel injections (a cumulative dose of 8 mg/kg, i. p.) induced cold and mechanical allodynia from days 10 to 21 in mice, and the i. p. administration of 0.025 mg/kg of cucurbitacins B and D attenuated both allodynia types. However, as cucurbitacin B showed a more toxic effect on non-cancerous (RAW 264.7) cells, further experiments were conducted with cucurbitacin D. The cucurbitacin D dose-dependently (0.025, 0.1, and 0.5 mg/kg) attenuated both allodynia types. In the spinal cord, paclitaxel injection increased the gene expression of noradrenergic (α 1-and α 2-adrenergic) receptors but not serotonergic (5-HT1A and 3) receptors. Cucurbitacin D treatment significantly decreased the spinal α 1- but not α 2-adrenergic receptors, and the amount of spinal noradrenaline was also downregulated. However, the tyrosine hydroxylase expression measured via liquid chromatography in the locus coeruleus did not decrease significantly. Finally, cucurbitacin D treatment did not lower the anticancer effect of chemotherapeutic drugs when co-administered with paclitaxel in CT-26 cell-implanted mice. Altogether, these results suggest that cucurbitacin D could be considered a treatment option against paclitaxel-induced neuropathic pain.

Anti-HIV Activity of Cucurbitacin-D against Cigarette Smoke Condensate-Induced HIV Replication in the U1 Macrophages.

Chemodietary agents are emerging as promising adjuvant therapies in treating various disease conditions. However, there are no adjuvant therapies available to minimize the neurotoxicity of currently existing antiretroviral drugs (ARVs). In this study, we investigated the anti-HIV effect of a chemodietary agent, Cucurbitacin-D (Cur-D), in HIV-infected macrophages using an in-vitro blood-brain barrier (BBB) model. Since tobacco smoking is prevalent in the HIV population, and it exacerbates HIV replication, we also tested the effect of Cur-D against cigarette smoke condensate (CSC)-induced HIV replication. Our results showed that Cur-D treatment reduces the viral load in a dose-dependent (0-1 µM) manner without causing significant toxicity at <1 µM concentration. Further, a daily dose of Cur-D (0.1 µM) not only reduced p24 in control conditions, but also reduced CSC (10 µg/mL)-induced p24 in U1 cells. Similarly, Cur-D (single dose of 0.4 µM) significantly reduced the CSC (single dose of 40 µg/mL)-induced HIV replication across the BBB model. In addition, treatment with Cur-D reduced the level of pro-inflammatory cytokine IL-1ß. Therefore, Cur-D, as an adjuvant therapy, may be used not only to suppress HIV in the brain, but also to reduce the CNS toxicity of currently existing ARVs.

HPMA-Based Copolymers Carrying STAT3 Inhibitor Cucurbitacin-D as Stimulus-Sensitive Nanomedicines for Oncotherapy.

The study describes the synthesis, physicochemical properties, and biological evaluation of polymer therapeutics based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers intended for a tumor-targeted immuno-oncotherapy. Water-soluble linear and cholesterol-containing HPMA precursors were synthesized using controlled reversible addition-fragmentation chain transfer polymerization to reach molecular weight Mn about 2 × 104 g·mol-1 and low dispersity. These linear or self-assembled micellar conjugates, containing immunomodulatory agent cucurbitacin-D (CuD) or the anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond, showed a hydrodynamic size of 10-30 nm in aqueous solutions. The CuD-containing conjugates were stable in conditions mimicking blood. Importantly, a massive release of active CuD in buffer mimicking the acidic tumor environment was observed. In vitro, both the linear (LP-CuD) and the micellar (MP-CuD) conjugates carrying CuD showed cytostatic/cytotoxic activity against several cancer cell lines. In a murine metastatic and difficult-to-treat 4T1 mammary carcinoma, only LP-CuD showed an anticancer effect. Indeed, the co-treatment with Dox-containing micellar polymer conjugate and LP-CuD showed potentiation of the anticancer effect. The results indicate that the binding of CuD, characterized by prominent hydrophobic nature and low bioavailability, to the polymer carrier allows a safe and effective delivery. Therefore, the conjugate could serve as a potential component of immuno-oncotherapy schemes within the next preclinical evaluation.

Mitogen-activated protein kinases are involved in cucurbitacin D-induced antitumor effects on adult T-cell leukemia cells.

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

Cucurbitacin D Overcomes Gefitinib Resistance by Blocking EGF Binding to EGFR and Inducing Cell Death in NSCLCs.

In this study, the mechanism of the anticancer effect through which cucurbitacin D (CuD) can overcome gefitinib resistance in NSCLC was investigated. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay, and cell migration and growth were observed by wound healing and colony formation assays, respectively. Levels of EGFR family members, protein kinase B, extracellular signal-regulated kinase, poly(ADP-ribose) polymerase, and G2/M phase-related proteins were detected by Western blot analysis. Immunofluorescence analysis was used to detect the intracellular expression of p-EGFR. Induction of apoptosis and cell cycle arrest was measured by flow cytometry. Solid-phase binding assays were used to determine binding to the EGFR family. CuD inhibits the phosphorylation of EGFR in gefitinib-resistant NSCLC cells and induces cell death via cell cycle arrest and apoptosis. CuD treatment or EGFR knockdown also suppressed the growth of gefitinib-resistant NSCLC cells. In addition, CuD overcame resistance by blocking EGF binding to EGFR in gefitinib-resistant NSCLC cells. In conclusion, we demonstrate that CuD overcomes gefitinib resistance by reducing the activation of EGFR-mediated survival in NSCLC and by inhibiting the combination of EGF and EGFR.

Synergistic anticancer effect of combined use of Trichosanthes kirilowii with cisplatin and pemetrexed enhances apoptosis of H1299 non-small-cell lung cancer cells via modulation of ErbB3.

BACKGROUND: Lung cancer is one of the most common malignancies worldwide. To treat lung cancer, various anticancer drugs were developed and tested, but they failed because of drug resistance. In the present study, we tested herbal medicines, such as TK and CuD, as anticancer drugs to decrease side effects and resistance. METHODS: Cell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by an annexin V-FITC/PI assay. We performed RTK kit analysis. Levels of p-ErbB3, p-STAT3, p-NF-κB, and caspases were measured by western blot analysis. Nuclear staining of ErbB3 was measured by immunocytochemistry. Transcriptional activity of STAT3 and NF-κB was detected by STAT3 and NF-κB luciferase reporter gene assays. RESULTS: We found a synergistic effect of TK with CDDP and PXD in primary culture of human NSCLC tumor cells. The combination of CDDP/PXD and TK or CuD inhibited the proliferation of H1299 cells. The combination of CDDP/PXD and TK or CuD induced sub-G1 and G2/M cell cycle arrest in H1299 cells. The combination of CDDP/PXD and TK or CuD induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in H1299 cells. We found that TK suppresses p-ErbB3 expression and signaling. The combination of CDDP/PXD and TK or CuD inhibited p-AKT, p-Erk, and p-JNK signaling and suppressed Stat3 and NF-κB transcriptional activity in H1299 cells. More importantly, the combination of CDDP/PXD and TK or CuD inhibited p-ErbB3 and downstream molecules in H1299 cells. The combination of CDDP/PXD and TK or CuD inhibited ErbB2/ErbB3 dimerization. Our results clearly demonstrate that the synergistic effect of CDDP/PXD and TK or CuD inhibits cell growth and induces apoptosis by inhibiting ErbB3 signaling. CONCLUSION: The combination of CDDP/PXD and TK or CuD decreases cell proliferation and induces apoptosis by inhibiting ErbB3 signaling in H1299 lung cancer cells. TK or CuD could be useful as a compound to treat lung cancer. Additionally, targeting ErbB3 may also be useful for treating lung cancer.

Cucurbitacin D Reprograms Glucose Metabolic Network in Prostate Cancer.

Prostate cancer (PrCa) metastasis is the major cause of mortality and morbidity among men. Metastatic PrCa cells are typically adopted for aberrant glucose metabolism. Thus, chemophores that reprogram altered glucose metabolic machinery in cancer cells can be useful agent for the repression of PrCa metastasis. Herein, we report that cucurbitacin D (Cuc D) effectively inhibits glucose uptake and lactate production in metastatic PrCa cells via modulating glucose metabolism. This metabolic shift by Cuc D was correlated with decreased expression of GLUT1 by its direct binding as suggested by its proficient molecular docking (binding energy -8.5 kcal/mol). Cuc D treatment also altered the expression of key oncogenic proteins and miR-132 that are known to be involved in glucose metabolism. Cuc D (0.1 to 1 µM) treatment inhibited tumorigenic and metastatic potential of human PrCa cells via inducing apoptosis and cell cycle arrest in G2/M phase. Cuc D treatment also showed inhibition of tumor growth in PrCa xenograft mouse model with concomitant decrease in the expression of GLUT1, PCNA and restoration of miR-132. These results suggest that Cuc D is a novel modulator of glucose metabolism and could be a promising therapeutic modality for the attenuation of PrCa metastasis.

Novel Mechanistic Insight into the Anticancer Activity of Cucurbitacin D against Pancreatic Cancer (Cuc D Attenuates Pancreatic Cancer).

Pancreatic cancer (PanCa) is one of the leading causes of death from cancer in the United States. The current standard treatment for pancreatic cancer is gemcitabine, but its success is poor due to the emergence of drug resistance. Natural products have been widely investigated as potential candidates in cancer therapies, and cucurbitacin D (Cuc D) has shown excellent anticancer properties in various models. However, there is no report on the therapeutic effect of Cuc D in PanCa. In the present study, we investigated the effects of the Cuc D on PanCa cells in vitro and in vivo. Cuc D inhibited the viability of PanCa cells in a dose and time dependent manner, as evident by MTS assays. Furthermore, Cuc D treatment suppressed the colony formation, arrest cell cycle, and decreased the invasion and migration of PanCa cells. Notably, our findings suggest that mucin 13 (MUC13) is down-regulated upon Cuc D treatment, as demonstrated by Western blot and qPCR analyses. Furthermore, we report that the treatment with Cuc D restores miR-145 expression in PanCa cells/tissues. Cuc D treatment suppresses the proliferation of gemcitabine resistant PanCa cells and inhibits RRM1/2 expression. Treatment with Cuc D effectively inhibited the growth of xenograft tumors. Taken together, Cuc D could be utilized as a novel therapeutic agents for the treatment/sensitization of PanCa.

Relationship between triterpenoid anticancer drug resistance, autophagy, and caspase-1 in adult T-cell leukemia.

We previously reported that the inflammasome inhibitor cucurbitacin D (CuD) induces apoptosis in human leukemia cell lines. Here, we investigated the effects of CuD and a B-cell lymphoma extra-large (Bcl-xL) inhibitor on autophagy in peripheral blood lymphocytes (PBL) isolated from adult T-cell leukemia (ATL) patients. CuD induced PBL cell death in patients but not in healthy donors. This effect was not significantly inhibited by treatment with rapamycin or 3-methyladenine (3-MA). The Bcl-xL inhibitor Z36 induced death in primary cells from ATL patients including that induced by CuD treatment, effects that were partly inhibited by 3-MA. Similarly, cell death induced by the steroid prednisolone was enhanced in the presence of Z36. A western blot analysis revealed that Z36 also promoted CuD-induced poly(ADP ribose) polymerase cleavage. Interestingly, the effects of CuD and Z36 were attenuated in primary ATL patient cells obtained upon recurrence after umbilical cord blood transplantation, as compared to those obtained before chemotherapy. Furthermore, cells from this patient expressed a high level of caspase-1, and treatment with caspase-1 inhibitor-enhanced CuD-induced cell death. Taken together, these results suggest that rescue from resistance to steroid drugs can enhance chemotherapy, and that caspase-1 is a good marker for drug resistance in ATL patients.

Autophagy is associated with cucurbitacin D-induced apoptosis in human T cell leukemia cells.

We previously reported that the inflammasome inhibitor cucurbitacin D (CuD) induces apoptosis in human leukemia cell lines. In the present study, we investigated the effects of co-treatment with an additional Bcl-xL inhibitor, Z36. Treatment with Z36 induced cell death in leukemia cell lines, with MT-4 cells exhibiting the lowest sensitivity to Z36. Co-treatment of cells with Z36 and CuD resulted in a greater degree of cell death for Hut78 and Jurkat cells than treatment with CuD alone. In contrast, co-treatment of MT-4 cells with Z36 and CuD had a suppressive effect on cell death. The autophagy inhibitor 3-methyladenine (3-MA) suppressed the growth of leukemia cell lines HuT78, Jurkat, MT-1, and MT-4. CuD-induced cell death was enhanced by 3-MA in Jurkat cells, but inhibited in MT-4 cells. Western blotting results revealed cleavage of poly(ADP ribose) polymerase (PARP), supporting CuD-induced cell death; 3-MA enhanced CuD-Z36-induced PARP cleavage. Taken together, our results indicate that autophagy negatively regulates chemical-induced cell death of leukemia cells, and that controlling autophagy could be beneficial in the development of more effective chemotherapies against leukemia.

Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells.

Breast cancer is the most common cancer for women and is a major cause of mortality in women. Doxorubicin is a generally used chemotherapy drug for breast cancer. However, multidrug resistance of breast cancer interferes with the chemotherapy. We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells. Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by Western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by Annexin V-FITC/propidium iodide assay. More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, and G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus. Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

Cucurbitacin-D-induced CDK1 mRNA up-regulation causes proliferation arrest of a non-small cell lung carcinoma cell line (NSCLC-N6).

Despite progress in chemotherapeutic agents, non-small cell lung cancers (NSCLC) still have a poor survival rate. Thus, development of new therapeutic strategies, specifically against cancer cells is still required. For this purpose, we treated the non-small cell lung cancer cell line NSCLC-N6 with the natural product cucurbitacin D (CucD) - extracted from the plant Ecballium elaterium in order first to assess its in vitro cytotoxicity, but also to study the genetic changes that it could bring out. CucD has shown a blocking in the G1 phase of the cell cycle in NSCLC-N6 cells prior to apoptotic cell death. The reverse transcriptase-polymerase chain reaction-differential display (RT-PCR-DD) technique was also applied on treated cells to elucidate the genetic mechanisms involved. We revealed an overexpression of Cyclin-dependent kinase 1 (CDK1) mRNA after treatment and, with the use of antisense oligonucleotides, an effective role in the proliferation arrest of NSCLC-N6 cells. The present study provides new insights about the mechanisms of proliferation arrest in tumor cells and open new ways of treatment to target tumor growth.

Morphological and physicochemical characterization of liposomes loading cucurbitacin E, an anti-proliferative natural tetracyclic triterpene.

Cucurbitacin E (Cuc E), an oxygenated triterpene molecule, has demonstrated anti-proliferative effect on various cancer cells. Here, we examined the effect of Cuc E on the membrane morphology and properties using differential scanning calorimetry, transmission electron microscopy and atomic force microscopy techniques. Dipalmitoylphosphatidylcholine vesicles were prepared by the thin film hydration method in the absence and presence of Cuc E at molar ratios 100:12 and 100:20. The loading efficiency of Cuc E was found to be higher than 98% upon HPLC analysis. The thermodynamic parameters suggest that Cuc E does not penetrate into the bilayers and interacts with the polar/apolar interface of the lipid membranes. Blank and Cuc E loaded liposomes prepared from a mixture of DPPC/DPPE/DPPG/Cho were imaged by TEM and AFM. Images obtained by TEM revealed unilamellar liposomes for blank and Cuc E loaded liposomes. AFM images showed that the size and the height of Cuc E loaded liposomes were respectively smaller and higher than blank ones. Results suggest that Cuc E produces modifications in the lipid membrane structures.

Cucurbitacin D is a new inflammasome activator in macrophages.

We previously reported that cucurbitacin D isolated from Trichosanthes kirilowii has anti-tumor roles to leukemia cells. However, the effect of cucurbitacin D on immune cells is not fully understood although there is no toxic activity to normal cells. In this study, immunomodulating activities of cucurbitacin D were investigated in macrophages. Cucurbitacin D could increase LPS-induced interleukin (IL)-1ß production in culture supernatant of THP-1 cells, peritoneal exudate cells (PECs), bone marrow derived macrophages (BMDMs), and RAW264 cells. At the transcriptional level, cucurbitacin D enhanced LPS-induced IL-1ß mRNA expression through activation of ERK1/2 mitogen-activated protein kinases (MAPKs). At the posttranscriptional level, the activation of caspase-1 induced by cucurbitacin D has also been demonstrated following treatment with a caspase-1 inhibitor and siRNA. Importantly, cucurbitacin D has further been shown to induce inflammasome activation independent of ERK1/2 activation. Western blotting showed interaction of NOD-like receptor family, pyrin domain containing 3 (NALP3) and apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain (ASC), suggesting activation of the inflammasome and a possible reason for activation of caspase-1. Taken together, these results suggest that cucurbitacin D could initiate immunomodulating activity in macrophages to lead to inflammasome activation as well as enhancement of LPS signaling.