p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver.

Background: Clinically, the persistence of HBV cccDNA is the major obstacle in anti-HBV therapy. However, the underlying mechanism of HBV cccDNA is poorly understood. The transcriptional factor STAT3 is able to activate HBV replication in liver. Approach & Results: RNA-Seq analysis demonstrated that cucurbitacin I targeting STAT3 was associated with virus replication in liver. HBV-infected human liver chimeric mouse model and HBV hydrodynamic injection mouse model were established. Then, we validated that cucurbitacin I effectively limited the stability of HBV cccDNA and HBV replication in cells, in which cucurbitacin I enhanced the sensitivity of pegylated interferon α (PEG-IFN α) against HBV via combination in vitro and in vivo. Mechanistically, we identified that cucurbitacin I increased the levels of APOBEC3B to control HBV cccDNA by inhibiting p-STAT3 in cells, resulting in the inhibition of HBV replication. Moreover, RNA-Seq data showed that E3 ubiquitin ligase DTX4 might be involved in the events. Then, we observed that HBV particles could upregulate DTX4 by increasing the levels of p-STAT3 in vitro and in vivo. The p-STAT3-elevated DTX4/male-specific lethal 2 (MSL2) independently and synergistically enhanced the stability of HBV cccDNA by facilitating the ubiquitination degradation of APOBEC3B in cells, leading to the HBV replication. Conclusions: p-STAT3-elevated DTX4 confers the stability of HBV cccDNA and HBV replication by facilitating the ubiquitination degradation of APOBEC3B. Cucurbitacin Ⅰ effectively enhances the sensitivity of PEG-IFN α in anti-HBV therapy by inhibiting the p-STAT3/DTX4/MSL2/APOBEC3B signalling. Our finding provides new insights into the mechanism of HBV cccDNA. The p-STAT3 and DTX4/MSL2 might serve as the therapeutical targets of HBV cccDNA.

Cucurbitacin I exerts its anticancer effects by inducing cell cycle arrest via the KAT2a-ube2C/E2F1 pathway and inhibiting HepG2-induced macrophage M2 polarization.

Hepatocellular carcinoma (HCC) is one of the most common malignancies globally, particularly prevalent in China, where it accounts for nearly half of the world's new cases and deaths each year, but has limited therapeutic options. This study systematically investigated the impact of cucurbitacin I on HCC cell lines including SK-Hep-1, Huh-7, and HepG2. The results revealed that cucurbitacin I not only inhibited cell proliferation, cell migration and colony formation, but also induced apoptosis in HCC cells. The apoptotic induction was accompanied by a decrease in the expression of the anti-apoptotic factor B-cell lymphoma 2 (Bcl2), and an elevation in the expression levels of pro-apoptotic factors, including tumor protein p53 (P53), bcl2 associated X-apoptosis regulator (Bax), and caspase3 (Cas3). Additionally, cucurbitacin I caused cell cycle arrest by modulating the lysine acetyltransferase 2A (KAT2A)-E2F transcription factor 1 (E2F1)/Ubiquitin-conjugating enzyme E2 C (UBE2C) signaling axis. In terms of regulation on tumor microenvironment, cucurbitacin I was demonstrated the ability to inhibit HCC cell-induced M2 polarization of macrophages. This comprehensive study unveils the multifaceted anti-cancer mechanisms of cucurbitacin I, providing robust support for its potential application in the treatment of HCC, offering new avenues for the future development of HCC treatment strategies.

Depolymerization of actin filaments by Cucurbitacin I through binding G-actin.

Cucurbitacins have high economic value as they are a major source of food and have pharmacological properties. Cucurbitacin I (CuI) is a plant-derived natural tetracyclic triterpenoid compound that shows an anticancer effect via inhibiting the JAK2-STAT3 signaling pathway. The actin cytoskeleton is the most abundant protein in cells and regulates critical events through reorganization in cells. In this study, it is aimed at determining the direct effect of CuI on actin dynamics. The fluorescence profile of G-actin in the presence of CuI (1-200 nM) shifted to a higher temperature, suggesting that G-actin binds CuI and that G-actin-CuI is more thermally stable than the ligand-free form. CuI dose-dependently inhibited the polymerization of F-actin in vitro and disrupted actin filaments in endothelial cells. Docking and MD simulations suggested that CuI binds to the binding site formed by residues I136, I175, D154, and A138 that are at the interface of monomers in F-actin. The migration ability of cells treated with CuI for 24 h was significantly lower than the control group (p < .001). This study reveals the molecular mechanisms of CuI in the regulation of actin dynamics by binding G-actin. More importantly, this study indicates a novel role of CuI as an actin-targeting drug by binding directly to G-actin and may contribute to the mode of action of CuI on anticancer activities.

Cucurbitacin I Reverses Tumor-Associated Macrophage Polarization to Affect Cancer Cell Metastasis.

The tumor microenvironment plays a critical role in tumor progression and immune regulation. As one of the most important components of the tumor microenvironment, macrophages have become a new therapeutic target for inhibiting tumor progression. Despite the well-documented anticancer activity of cucurbitacin I, its effect on macrophages remains unclear. In this study, we established a coculture system of macrophages and cancer cells under hypoxic conditions to simulate the tumor-promoting environment mediated by M2-like macrophages. We determined whether cucurbitacin I modulates M2-like polarization in macrophages in vitro and conducted RNA sequencing to identify gene expression changes induced by cucurbitacin I in macrophages. The results indicated a remarkable inhibition of the M2-like polarization phenotype in macrophages following treatment with cucurbitacin I, which was accompanied by the significant downregulation of heme oxygenase-1. Moreover, we found that cucurbitacin I-treated macrophages reduced the migration of cancer cells by inhibiting the M2 polarization in vitro. These findings highlight the potential of cucurbitacin I as a therapeutic agent that targets M2-like macrophages to inhibit cancer cell metastasis. Our study provides novel insights into the intricate interplay among macrophage polarization, cucurbitacin I, and heme oxygenase-1, thereby opening new avenues for cancer treatment.

JSI-124 Induces Cell Cycle Arrest and Regulates the Apoptosis in Glioblastoma Cells.

Cucurbitacin I (JSI-124), derived from Cucurbitaceae, has shown the potential to induce apoptosis and cell cycle arrest in some cancer cells. However, the effect of JSI-124 on glioblastoma multiforme (GBM) cell cycle and apoptosis is still unclear. Our investigation revealed that JSI-124 effectively reduced cell viability in GBM cells, leading to apoptosis and increased caspase-3 activity. Intriguingly, JSI-124 caused the accumulation of G2/M phase to regulate cell cycle, confirmed by MPM-2 staining and increased protein synthesis during mitosis by mitotic index analysis. Western blot analysis found that JSI-124 affected the progression of G2/M arrest by downregulating the CDK1 and upregulating the cyclinB1, suggesting that JSI-124 disrupted the formation and function of the cyclin B1/CDK1 complex in GBM8401 and U87MG cells. However, we found the JSI-124-regulated cell cycle G2/M and apoptosis-relative gene in GBM8401 and U87MG cells by NGS data analysis. Notably, we found that the GBM8401 and U87MG cells observed regulation of apoptosis and cell-cycle-related signaling pathways. Taken together, JSI-124 exhibited the ability to induce G2/M arrest, effectively arresting the cell cycle at critical stages. This arrest is accompanied by the initiation of apoptosis, highlighting the dual mechanism of action of JSI-124. Collectively, our findings emphasize that JSI-124 holds potential as a therapeutic agent for GBM by impeding cell cycle progression, inhibiting cell proliferation, and promoting apoptosis. As demonstrated by our in vitro experiments, these effects are mediated through modulation of key molecular targets.

The curative effect of some natural active compounds for liver cancer.

Cancer is a disease that has become widespread recently and has been studied extensively. It is of great importance to find an active and effective treatment quickly due to the emergence of the disease and its spread to many tissues in the organism by metastasis. In this study, it is aimed to detect active and active substances that are highly effective on cancer cells in a short time by using docking scores, the accuracy of which has been proven by many studies. Today, many medicinal plants are being studied for therapeutic purposes. In this study, the activities of the prominent active substances in these medicinal plants were compared with the docking scores and the molecules with the highest inhibition effect on liver cancer receptors were determined. The data obtained in this study are of great importance in terms of guiding experimental studies by detecting active substances effective on liver cancer by preventing time and material loss. Considering the results obtained from this study, it can be concluded that Cucurbitacin I and Cucurbitacin E, Thymol, Piperine, and Carvacrol are very effective for the inhibition of liver cancer cell receptors.

Cucurbitacin I exhibits anticancer efficacy through induction of apoptosis and modulation of JAK/STAT3, MAPK/ERK, and AKT/mTOR signaling pathways in HepG2 cell line.

Hepatocellular carcinoma is a common cancer type, especially among men. Although cucurbitacin I (CuI), widely found in plants belonging to the Ecballium elaterium (E. L) plant family, has been shown to have antitumorigenic properties in many cancer types, its anticancer effect, molecular mechanism, and apoptotic effect mediated by signal pathways on hepatocellular carcinoma have not been fully clarified. In the present study, we investigated the anticancer effect of CuI treated at different doses on the HepG2 cell line and the underlying mechanism in vitro. High-purity CuI was obtained from the E. elaterium plant with the aid of HPLC. The effects of this substance on the viability of cells were studied by the MTT assay. The effects of CuI on cell cycle progression and apoptosis were studied with flow cytometry. DNA breaks were analyzed by the Comet assay method. The proteins and genes involved in the JAK/STAT3, MAPK/ERK, and AKT/mTOR signaling pathways were investigated using Western blot and qRT-PCR, respectively. The results of this study demonstrated that CuI significantly reduced HepG2 cell growth in vitro, induced antiproliferation, and G2/M phase of the cell cycle was interrupted. PRACTICAL APPLICATIONS: CuI administration was shown to downregulate the levels of proteins in the PI3K/AKT/mTOR, MAPK, and JAK2/STAT3 cascades in HepG2 cells. CuI also reduced the expression of MAPK, STAT3, mTOR, JAK2, and Akt genes in different concentrations. DNA breaks are formed as a result of this effect. CuI, by reducing cell proliferation and promoting apoptosis, was found to have potential as a chemotherapeutic agent of hepatocellular carcinoma.

Cucurbitacin I inhibits the proliferation of pancreatic cancer through the JAK2/STAT3 signalling pathway in vivo and in vitro.

Pancreatic cancer is one of the most aggressive solid malignancies, as it has a 5-year survival rate of less than 10%. The growth and invasion of pancreatic cancer cells into normal tissues and organs make resection and treatment difficult. Finding an effective chemotherapy drug for this disease is crucial. In this study, we selected the tetracyclic triterpenoid compound cucurbitacin I, which may be used as a potential therapeutic drug for treating pancreatic cancer. First, we found that cucurbitacin I inhibited pancreatic cancer proliferation in a dose-time dependent manner. Further studies have shown that cucurbitacin I blocks the cell cycle of pancreatic cancer in the G2/M phase and induces cell apoptosis. In addition, under the action of the compound, the invasion ability of cells was greatly reduced and markedly impaired the growth of pancreatic tumour xenografts in nude mice. Furthermore, the decrease in pancreatic cancer cell proliferation caused by cucurbitacin I appeared to involve JAK2/STAT3 signalling pathway inhibition, and the use of JAK2/STAT3 activators effectively restored the inhibition. In conclusion, our research may provide a basis for the further development of pancreatic cancer treatment drugs.

Neuroprotective potential of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element signaling modulator cucurbitacin I upon glucose and oxygen deprivation/reperfusion (OGD/RP).

This study aimed to investigate the inhibitory effect and mechanism of Cucurbitacin I (Cu I) on apoptosis, oxidative stress, and mitophagy in PC12 cells with glucose and oxygen deprivation/reperfusion (OGD/RP) injury. OGD/RP cell injury model was established by gas anoxic cell incubator and glucose-free medium. The cells were divided into the control group, OGD/RP group, OGD/RP + Cu I group, and OGD/RP + Cu I + 2 µM nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 group. The results showed that apoptotic rate and reactive oxygen species (ROS) production were significantly increased in OGD/RP group, which were reversed by Cu I pretreatment. Meanwhile, western blot analysis proved that Cu I inhibited OGD/RP-induced mitophagy, manifested as the decreased expression of PTEN-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin-protein ligase (Parkin), and light chain 3 (LC3) Ⅱ∕LC3 I, as well as the increased expression of P62. Furthermore, immunofluorescence (IF) staining showed that Cu I reduced the co-localized puncta of LC3 with TOM20 in OGD/RP-induced PC12 cells. Similarly, transmission electron microscope finding is consistent with the IF results. Mechanically, after Cu I and OGD/RP treatments, nuclear Nrf2 expression and the levels of downstream target genes were significantly upregulated compared with OGD/RP alone treatment. Nrf2 inhibition reversed the protective effects of Cu I on OGD/RP-induced injury in PC12 cells. The present study provides evidence of the neuroprotective effect of Cu I unraveling its potential as a potential therapeutic candidate for the treatment of ischemic stroke.

Cucurbitacin I induces apoptosis in ovarian cancer cells through oxidative stress and the p190B­Rac1 signaling axis.

Ovarian cancer is a serious threat to women's life and health, with a high mortality rate. Therefore, in addition to improving surgery for ovarian cancer, it is particularly important to develop novel drug treatments. In the present study, the anticancer effects of cucurbitacin I, a natural product, were investigated. Cucurbitacin I impaired the viability of SKVO3 cells in a concentration­ and time­dependent manner. Apoptosis was involved in the process of cucurbitacin I­induced cell death, with an increase observed in cleaved­caspase 3 and BAX, and a decrease in Bcl­2. Cucurbitacin I caused a notable increase in intracellular reactive oxygen species, and regulated Kelch­like ECH­associated protein 1 and nuclear factor erythroid­derived 2­like 2 to decrease the expression of antioxidant­related genes. In addition, Cucurbitacin I induced cell shrinkage by regulating the p190BRhoGAP (p190B)­Rac1 signaling axis related to the cytoskeleton. In brief, these results suggested that cucurbitacin I induced cell death through oxidative stress and the p190B­Rac1 signaling axis in SKVO3 cells. The results may provide novel evidence for the treatment of ovarian cancer.

Elaeocarpus reticulatus fruit extracts reduce viability and induce apoptosis in pancreatic cancer cells in vitro.

Treatment options for pancreatic cancer (PC) are severely limited due to late diagnosis, early metastasis and the inadequacy of chemotherapy and radiotherapy to combat the aggressive biology of the disease. In recent years, plant-derived bioactive compounds have emerged as a source of novel, anti-cancer agents. Used in traditional medicine worldwide, Elaeocarpus species have reported anti-inflammatory, antioxidant and anti-cancer properties. This study aimed to isolate and identify potential anti-PC compounds in the fruit of Elaeocarpus reticulatus Sm. A 50% acetone crude extract significantly decreased the viability of four pancreatic cell lines (≥ 10 µg/mL for BxPC-3 cells) and induced apoptosis in BxPC-3 and HPDE cells. Analysis by HPLC identified the triterpenoid Cucurbitacin I as a likely component of the extract. Furthermore, treatment with Cucurbitacin I significantly reduced the viability of HPDE and BxPC-3 cells, with results comparable to the same concentration of gemcitabine. Interestingly, attempts to isolate bioactive compounds revealed that the crude extract was more effective at reducing PC-cell viability than the fractionated extracts. This study provides initial insight into the bioactive constituents of E. reticulatus fruits.

[Simultaneous determination of cucurbitacin B, E and I in plasma, urine, and melon and fruit vegetables by ultra performance liquid chromatography-triple quadrupole mass spectrometry using atmospheric pressure chemical ionization].

A method for the determination of cucurbitacin B (CuB), cucurbitacin I (CuI) and cucurbitacin E (CuE) in plasma, urine and melon and fruit vegetables was developed by ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS). The target analytes in plasma and urine were extracted and cleaned-up by solid supported liquid-liquid extraction, while those in melon and fruit vegetables were extracted with acetonitrile and then diluted with water. CuB, CuI and CuE were separated on an XBridge BEH C18 column (100 mm×3.0 mm, 2.5 µm) with gradient elution using mobile phases of methanol and 0.025% (v/v) ammonia aqueous solution. An atmospheric pressure chemical ionization interface was used as the ion source and the analysis was performed in negative ionization multiple reaction monitoring (MRM) mode. The cucurbitacins in plasma and urine were quantified by the matrix working standard curve internal standard method, while those in melon and fruit vegetables were quantified by the solvent standard curve external standard method. Oleandrin was used as the internal standard. The average recoveries were 89.0%-113% for the three cucurbitacins in plasma and urine, with RSDs of 1.7%-12.2% (n=6). The average recoveries were 87.6%-114% for the three cucurbitacins in melon and fruit vegetables, with RSDs of 4.1%-11.1% (n=6). The limit of detection (S/N=3) of the three cucurbitacins was 0.03 µg/L in plasma and urine, and 5-10 µg/kg in melon and fruit vegetables. The method is simple, sensitive and accurate. It has been used for the determination of cucurbitacins in bitter bottle gourd and in the plasma and urine of patients poisoned by bitter bottle gourd, CuB was successfully detected.

Cucurbitacin I induces cancer cell death through the endoplasmic reticulum stress pathway.

Endoplasmic reticulum stress (ERS) is usually involved in tumor development and progression, and anticancer agents have recently been recognized to induce ERS. Cucurbitacin-I showed a potent anticancer action by inducing apoptosis through the inhibition of signal transducer and activator of transcription 3 pathway and triggering autophagic cell death. It is not known whether ERS mediates the cancer cell death induced by cucurbitacin-I. Here, we investigated the role of ERS in cucurbitacin-I-treated SKOV3 ovarian cancer cells and PANC-1 pancreatic cancer cells. We confirmed that cucurbitacin-I caused cell death and stirred excessive ERS levels by activating inositol requiring enzyme 1α (IRE1α) and protein kinase R-like endoplasmic reticulum kinase (PERK), as well as PERK downstream factors, including IRE1α and C/EBP homologous protein, but not activating transcription factor 6 (ATF6α) pathway, which was in parallel with the increased Bax and caspase-12-dependent ERS-associated apoptosis, autophagy and autophagy flux levels and caspase-independent nonapoptotic cell death. Furthermore, 4-phenylbutyrate, an ERS inhibitor, suppressed cucurbitacin-I-induced apoptosis, autophagy, autophagy flux, and autophagic cell death. Simultaneously, there are positive correlations among ERS and cucurbitacin-I-induced reactive oxygen species and Ca 2+ . Our results suggested that cucurbitacin-I-induced cancer cell death through the excessive ERS and CHOP-Bax and caspase-12-dependent ERS-associated apoptosis, as well as ERS-dependent autophagy, autophagy flux, and caspase-independent nonapoptotic cell death. These novel signaling insights may be useful for developing new, effective anticancer strategies in oncotherapy.

Cucurbitacin I induces pro-death autophagy in A549 cells via the ERK-mTOR-STAT3 signaling pathway.

Natural products are a great source of cancer chemotherapeutic agents. In the present study, the anticancer effects of cucurbitacin I on A549 cells were investigated. Cucurbitacin I decreased cell viability, inhibited colony formation, and induced apoptosis in A549 cells. Cucurbitacin I caused accumulation of autophagosome and dose-dependent expression of LC3II protein. Autophagy inhibitors 3-methyladenine (3-MA) inhibited autophagy induced by cucurbitacin I and relieved cucurbitacin I-triggered cell death and apoptosis in A549 Cells. Cucurbitacin I treatment inhibits the ERK activation and the downstream phosphorylation level of mTOR and STAT3, but not the PI3K/Akt pathway. Furthermore, treatment with the mTOR activator MHY-1485, which also suppressed cucurbitacin I-induced LC3II expression, and also reversed cucurbitacin I-induced cell death and apoptosis. Taken together, these results suggest that cucurbitacin I induced pro-death autophagy through ERK/mTOR/STAT3 signaling cascade in A549 cells.

Development and validation of a quantification method for cucurbitacins E and I in rat plasma: Application to population pharmacokinetic studies.

Cucurbitacin E is a potential drug candidate due to its anticancer activity, recognition of its molecular targets, and synergism with other drugs used for cancer treatment. However, the use of cucurbitacin E in clinical practice is not possible because of important knowledge gaps in its preclinical and clinical pharmacokinetic characteristics. Cucurbitacin E is hydrolyzed to cucurbitacin I in plasma and in human liver microsomes. The aim of this study was to evaluate the population pharmacokinetics of cucurbitacin E and of its metabolite cucurbitacin I in rats. The method for the sequential analysis of cucurbitacins E and I in rat plasma was developed using LC-MS/MS. Plasma aliquots of 50µL were deproteinized with acetonitrile and clobazam was added as internal standard. The extracts were injected into an RP-18 column and eluted with a mobile phase consisting of a mixture of acetonitrile:water:methanol (32:35:33, v/v/v). The method was precise and accurate, showing linearity in the range of 1-100ng cucurbitacin E/mL plasma and of 0.4-200ng cucurbitacin I/mL plasma. The method was applied to the pharmacokinetic evaluation of cucurbitacin E administered intravenously to male Wistar rats (1mg/kg). Serial blood samples were collected up to 24h after administration. The plasma concentrations of cucurbitacin E were quantified up to 16h, while the plasma concentrations of cucurbitacin I remained below the limit of quantification. A population pharmacokinetic model was developed for cucurbitacin E using the NONMEM program, with adequate goodness of fit and predictive performance. The following pharmacokinetic parameters were obtained: release time of 0.45h, volume of distribution of 27.22L, clearance of 4.13L/h, and elimination half-life of 4.57h.

Cucurbitacin-I induces hypertrophy in H9c2 cardiomyoblasts through activation of autophagy via MEK/ERK1/2 signaling pathway.

Cucurbitacin-I, a natural triterpenoids initially identified in medicinal plants, shows a potent anticancer effect on a variety of cancer cell types. Nevertheless, the cardiotoxicity of cucurbitacin-I has not heretofore been reported. In this study, the mechanisms of cucurbitacin-I-induced cardiotoxicity were examined by investigating the role of MAPK-autophagy-dependent pathways. After being treated with 0.1-0.3µM cucurbitacin-I for 48h, H9c2 cells showed a gradual decrease in the cell viabilities, a gradual increase in cell size, and mRNA expression of ANP and BNP (cardiac hypertrophic markers). Cucurbitacin-I concentration-dependent apoptosis of H9c2 cells was also observed. The increased apoptosis of H9c2 cells was paralleling with the gradually strong autophagy levels. Furthermore, an autophagy inhibitor, 3-MA, was used to block the cucurbitacin-I-stirred autophagy, and then the hypertrophy and apoptosis induced by 0.3µM cucurbitacin-I were significantly attenuated. In addition, cucurbitacin-I exposure also activated the MAPK signaling pathways, including ERK1/2, JNK, and p38 kinases. Interestingly, only the ERK inhibitor U0126, but not the JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580, weakened the induction of 0.3µM cucurbitacin-I in hypertrophy, autophagy and apoptosis. Our findings suggest that cucurbitacin-I can increase the autophagy levels of H9c2 cells, most likely, through the activation of an ERK-autophagy dependent pathway, which results in the hypertrophy and apoptosis of cardiomyocytes.

Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.

Nanoprecipitation is more efficient than emulsion solvent evaporation method to encapsulate cucurbitacin I in PLGA nanoparticles.

Cucurbitacin I is a hydrophobic molecule that exerts a degree of polarity, which is expected to complicate its loading in PLGA nanoparticles by the classical emulsion solvent evaporation technique. In the current study, variants of emulsion solvent evaporation method were used to prepare PLGA nanoparticles of cucurbitacin: CI-NP1 (single emulsion starting with 1000 µg drug), CI-NP2 (double emulsion starting with 250 µg drug), and CI-NP3 (double emulsion starting with 500 µg drug). On the other hand, CI-NP4 was prepared by nanoprecipitation (starting with 1000 µg drug). In CI-NP1, cucurbitacin I encapsulation efficiency (EE) was 1.29%. The employment of double emulsion, in CI-NP2 and CI-NP3, increased cucurbitacin I EE to 4.8% and 7.96%, respectively. Nanoprecipitation significantly increased the EE of cucurbitacin I to 48.79% in CI-NP4. It is likely that cucurbitacin I escapes with the organic solvent after the emulsification step to the aqueous phase leading to ineffective entrapment in the polymeric matrix. Avoiding emulsification seems efficient in increasing cucurbitacin I disposition in the instantly-precipitating NPs. Therefore, nanoprecipitation method increases cucurbitacin I entrapment in PLGA NPs and possibly other water-insoluble polar drugs.

Cucurbitacin-I, a natural cell-permeable triterpenoid isolated from Cucurbitaceae, exerts potent anticancer effect in colon cancer.

Cucurbitacin-I is a triterpenoids found in medicinal plants and have diverse pharmacological and biological activities. In this study, the antitumor effects of cucurbitacin-I on colon cancer and possible roles in apoptosis and cell cycle arrest were investigated. Treatment of SW480 cells, a human colon cancer cells, with cucurbitacin-I decreased cell viability and cell proliferation in a concentration-dependent manner. Also, cucurbitacin-I induced G2/M phase cell cycle arrest in SW480 cells with a decreased expression of cell cycle proteins including cyclin B1, cyclin A, CDK1, and CDC25C. Moreover, cucurbitacin-I induced increased cleavage of caspase-3, -7, -8, -9, and poly ADP ribose polymerase. When we examined the inhibitory effect of cucurbitacin-I on tumor growth in vivo, cucurbitacin-I effectively inhibited the tumorigenicity and growth of CT-26 cells in syngenic BALB/c mice. In summary, the present study showed that cucurbitacin-I reduced colon cancer cell proliferation by enhancing apoptosis and causing cell cycle arrest at the G2/M phase.

Cucurbitacin I induces protective autophagy in glioblastoma in vitro and in vivo.

There is an urgent need for new therapeutic avenues to improve the outcome of patients with glioblastoma multiforme (GBM). Current studies have suggested that cucurbitacin I, a natural selective inhibitor of JAK2/STAT3, has a potent anticancer effect on a variety of cancer cell types. This study showed that autophagy and apoptosis were induced by cucurbitacin I. Exposure of GBM cells to cucurbitacin I resulted in pronounced apoptotic cell death through activating bcl-2 family proteins. Cells treatment with cucurbitacin I up-regulated Beclin 1 and triggered autophagosome formation and accumulation as well as conversion of LC3I to LC3II. Activation of the AMP-activated protein kinase/mammalian target of rapamycin/p70S6K pathway, but not the PI3K/AKT pathway, occurred in autophagy induced by cucurbitacin I, which was accompanied by decreased hypoxia-inducible factor 1α. Stable overexpression of hypoxia-inducible factor 1α induced by FG-4497 prevented cucurbitacin I-induced autophagy and down-regulation of bcl-2. Knockdown of beclin 1 or treatment with the autophagy inhibitor 3-methyladenine also inhibited autophagy induced by cucurbitacin I. A coimmunoprecipitation assay showed that the interaction of Bcl-2 and Beclin 1/hVps34 decreased markedly in cells treated with cucurbitacin I. Furthermore, knockdown of beclin 1 or treatment with the lysosome inhibitor chloroquine sensitized cancer cells to cucurbitacin I-induced apoptosis. Finally, a xenograft model provided additional evidence for the occurrence of cucurbitacin I-induced apoptosis and autophagy in vitro. Our findings provide new insights into the molecular mechanisms underlying cucurbitacin I-mediated GBM cell death and may provide an efficacious therapy for patients harboring GBM.