Cucurbitacin Research Articles

Abstract 2686: The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells

Abstract Glioblastoma is a lethal brain tumor. Despite aggressive treatment, the median survival time is 6 to 12 months. These tumors acquire resistance against many therapeutic regimens. There is a need to screen for potential new drugs and drug combinations. Cucurbitacin B (CuB), a compound obtained from plants, and evodiamine (Evo), a supplement, have shown promising results in various neoplasms but have not been well studied in glioblastoma and especially, as a combination treatment. This study investigated the combined effects of CuB and Evo on U-87MG human glioma cell line. Results demonstrated that CuB and Evo individually decreased the U-87MG cell viability in time and dose dependent manners. The effect was significantly pronounced with the combined treatment. The combination treatment of CuB and Evo induced cellular apoptosis most likely by interfering with the PI3K/AKT and JNK/ERK pathways. In addition, the flow cytometry analysis showed that the combination treatment arrested the cell cycle at the G2M phase and inhibited cell propagation. Moreover, CuB and Evo combined effect successfully inhibited cellular migration. The cytotoxic effect of combination treatment of CuB and Evo was three times lower in neuronal PC-12 cells compared to U-87MG cells. Overall, a combination of cucurbitacin B and evodiamine proved to be a superior potential anti-neoplastic treatment in U-87MG cells as compared to individual treatment, and may be considered as a potential adjuvant therapy for GBM. Citation Format: Vrutti V. Mehta, Kenza E. Benzeroual. The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2686.

Antitumor activity and mechanism of cucurbitacin B in A549/DDP cells.

Cucurbitacin B (CuB) is a class of tetracyclic triterpenoids isolated from Cucurbitaceae with a wide range of anti-inflammatory and anti-tumor activities, mainly used in hepatitis and hepatocellular carcinoma, while there is relatively little research and application of this drug for lung cancer. In this study, CuB was administered on A549/DDP cells to observe how it affected the cells and their mechanism of action. CuB demonstrated good anti-tumor activity against A549/DDP cells in a dose-dependent manner and caused changes in the hedgehog (Hh) pathway. The results showed that CuB greatly inhibits the proliferation and the invasion of A549/DDP cells, and promoted apoptosis of A549/DDP cells. Meanwhile, it changed the expression of p53-related genes at the RNA and protein level. In conclusion, this experiment provides a theoretical basis for new applications of CuB and new thoughts on the mechanism of its anti-tumor activity, and provides a direction for deep research.

Label-free-based quantitative proteomic analysis of the inhibition of cisplatin-resistant ovarian cancer cell proliferation by cucurbitacin B

BackgroundOvarian cancer is a serious threat to women's health, and resistance to chemotherapeutic drugs constitutes one of the principal reasons for ovarian cancer recurrence and the low overall survival rate. Therefore, it is of paramount importance to develop additional and more-effective drugs to combat resistance to chemotherapeutic drugs. Cucurbitacin B (CuB) is a natural compound found in food plants such as bitter gourd and pumpkin, and it manifests favorable antitumor effects on a variety of malignant tumors. PurposeThe present study aimed to determine the mechanism effects of CuB overcomes tumor-drug resistance in ovarian cancer. MethodsWe used CCK-8, Edu, flow cytometric assays and cisplatin-resistant ovarian cancer xenograft mouse model to evaluate the cellular proliferation, cellular apoptosis.and tumor growth. We subsequently applied a pharmacoproteomic approach to analyze the molecular mechanisms by which CuB inhibited the proliferation of cisplatin-resistant ovarian cancer cells. We also employed western blot and molecular docking experiments to verify elements of PI3K/Akt/mTOR pathway expression. ResultsWe found that CuB inhibited cellular proliferation and promoted apoptosis in cisplatin-resistant ovarian cancer cell lines. We discerned that CuB inhibited tumor growth of xenograft mouse tumors. We ascertained that treatment of A2780-DDP cells with CuB resulted in the differential expression of 305 proteins, with 202 proteins downregulated and 103 proteins upregulated. Of these proteins, the mTOR protein was significantly downregulated in the drug-treated group. We also found that CuB inhibited PI3K, Akt, and mTOR and that it activated cGAS expression upstream of PI3K and inhibited ATR expression. Molecular docking experiments revealed that CuB was hydrogen-bonded to mTOR proteins at Gly (2142) and Thr (2207), with a binding force of –10.2 kcal/mol. ConclusionOur study confirmed that cucurbitacin B inhibits the PI3K/Akt/mTOR signaling pathway, targets mTOR, suppresses the proliferation of cisplatin-resistant ovarian cancer cells.And we also found that cucurbitacin B induces DNA damage, activates cGASA and recruits IKBα,playing a crucial role in eliciting anti-tumor immunity. We herein uncovered a new use for CuB in inhibiting tumor-drug resistance, providing a novel approach to overcoming chemotherapeutic drug resistance in ovarian cancer.

Allosteric regulation of the lid domain of PCK2 as a novel strategy for modulating mitochondrial dynamics.

Aberrant PCK2 overexpression has been linked to an unfavorable prognosis and shorter survival, particularly in hepatocellular carcinoma (HCC). Thus, the inactivation of PCK2 provides a promising strategy for HCC treatment. In this study, we used a chemical genetic strategy to identify a natural-derived small-molecule cucurbitacin B (CuB) as a selective PCK2 inhibitor. CuB covalently bound to PCK2 at a unique Cys63 site, blocking the Ω-loop lid domain formation via a previously undisclosed allosteric mechanism. Additionally, targeted lipidomics analysis also revealed that CuB destroyed mitochondrial membrane integrity, leading to the disruption of mitochondrial fusion-fission dynamics. Taken together, this study highlights the discovery of a small-molecule CuB, which reprograms lipid metabolism for controlling mitochondrial dynamics via targeting PCK2 in cancer cells.

Okadaic Acid-Induced Alzheimer's in Rat Brain: Phytochemical Cucurbitacin E Contributes to Memory Gain by Reducing TAU Protein Accumulation.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive decline, with hallmark pathologies related to amyloid beta (Aβ) and TAU. Natural phytochemicals show promise for drug discovery to fill the current therapeutic innovation gap in AD. This study investigated the effect of cucurbitacin E (CuE), one of the bioactive components of Ecballium elaterium, on TAU fibril formation in okadaic acid-induced AD in rats. In a randomized design, we assigned 30 female Sprague Dawley rats to one of five experimental groups: (1) control, (2) stereotaxic surgery, (3) stereotaxic surgery + artificial cerebrospinal fluid, (4) stereotaxic surgery + okadaic acid (AD model), and (5) stereotaxic surgery + okadaic acid + CuE treatment. For experimental groups 4 and 5, rats were administered OKA-ICV (200 ng/kg) followed by CuE (4 mg/[kg·day], intraperitoneally) for 20 days. Expression of the MAPK1/3 and MAPK14 genes associated with TAU metabolism, hippocampal protein levels of these genes, cognitive functions of the rats, and histological accumulation of TAU in the brain were evaluated. Our findings in this preclinical model collectively suggest that phytochemical CuE contributes to memory gain by reducing TAU protein accumulation, which warrants further evaluation in future in vitro and in vivo studies.

Synergistic effect of cucurbitacin E and myricetin on Anti-Non-Small cell lung cancer: Molecular mechanism and therapeutic potential

BackgroundNon-small cell lung cancer (NSCLC) is associated with extremely high morbidity and mortality rates worldwide. Citrullus colocynthis (L.) Schrad, widely distributed in Asian and African countries, is used to treat cancers in traditional Uyghur medicine. Hypothesis/PurposeThe combination of Cucurbitacin E (CuE) and Myricetin (Myr) of C. colocynthis could treat NSCLC by targeting autophagy. Study DesignThe potential anti-cancer components (CuE and Myr) of C. colocynthis were identified using in-silico methods and further in vitro explored the anti-NSCLC properties of the combination of CuE and Myr. MethodsNetwork pharmacology and molecular docking were used to identify potential therapeutic compounds of C. colocynthis for the treatment of NSCLC. In A549 cells, the anti-cancer activities and synergy of CuE and Myr were studied using CompuSyn, their mechanism behind autophagy regulation was determined by western blotting and immunofluorescence staining. ResultsCuMy-12 (CuE: 0.5 µM, Myr: 20 µM), a combination of CuE and Myr from C. colocynthis, inhibited A549 cell proliferation and colony formation, and induced apoptosis and cell cycle arrest in the G0/G1 phase, exhibiting a synergistic effect. Furthermore, CuMy-12 inhibited autophagy and activation of the PI3K/AKT/mTOR signaling pathway, which was characterized by a decrease in Beclin 1, AKT, and phospho-AKT proteins. ConclusionCuMy-12 can be considered a natural candidate with anticancer activity for autophagy-based regulation, but mechanistic and clinical studies are required to validate its potential.

Metabolomics profiling of AKT/c-Met-induced hepatocellular carcinogenesis and the inhibitory effect of Cucurbitacin B in mice.

Hepatocellular carcinoma (HCC), the most common kind of liver cancer, accounts for the majority of liver cancer diagnoses and fatalities. Clinical aggressiveness, resistance to traditional therapy, and a high mortality rate are all features of this disease. Our previous studies have shown that co-activation of AKT and c-Met induces HCC development, which is the malignant biological feature of human HCC. Cucurbitacin B (CuB), a naturally occurring tetracyclic triterpenoid compound with potential antitumor activity. However, the metabolic mechanism of AKT/c-Met-induced Hepatocellular Carcinogenesis and CuB in HCC remains unclear. In this study, we established an HCC mouse model by hydrodynamically transfecting active AKT and c-Met proto-oncogenes. Based on the results of hematoxylin-eosin (H&E), oil red O (ORO) staining, and immunohistochemistry (IHC), HCC progression was divided into two stages: the early stage of HCC (3weeks after AKT/c-Met injection) and the formative stage of HCC (6weeks after AKT/c-Met injection), and the therapeutic effect of CuB was evaluated. Through UPLC-Q-TOF-MS/MS metabolomics, a total of 26 distinct metabolites were found in the early stage of HCC for serum samples, while in the formative stage of HCC, 36 distinct metabolites were found in serum samples, and 13 different metabolites were detected in liver samples. 33 metabolites in serum samples and 11 in live samples were affected by CuB administration. Additionally, metabolic pathways and western blotting analysis revealed that CuB influences lipid metabolism, amino acid metabolism, and glucose metabolism by altering the AKT/mTORC1 signaling pathway, hence decreasing tumor progression. This study provides a metabolic basis for the early diagnosis, therapy, and prognosis of HCC and the clinical application of CuB in HCC.

Prophylactic effects of cucurbitacin B in the EAE Model of multiple sclerosis by adjustment of STAT3/IL-23/IL-17 axis and improvement of neuropsychological symptoms.

Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system. Although remarkable progress has been made in treating MS, current therapies are less effective in protecting against the progression of the disease. Since cucurbitacins have shown an extreme range of pharmacological properties, in this study, we aimed to investigate the prophylactic effect of cucurbitacin B (CuB) in the experimental MS model. Experimental autoimmune encephalomyelitis (EAE) induced by subcutaneous immunization of MOG35-55 in C57BL/6 mice. CuB interventions (0.5 and 1mg/kg, i.p.) were performed every other day from the first day of EAE induction. Assessment of clinical scores and motor function, inflammatory responses, and microglial activation were assessed by qRT-PCR, western blotting, and immunohistochemical (IHC) analyses. CuB (1mg/kg) significantly decreased the population of CD45+ (P < 0.01), CD11b+ (P < 0.01) and CD45+/CD11b+ (P < 0.05) cells in cortical lesions of EAE mice. In addition, activation of STAT3 (P < 0.001), expression of IL-17A and IL-23A (both mRNA and protein), and transcription of Iba-1 significantly decreased. On the contrary, CuB (1mg/kg) significantly increased the transcription of MBP and Olig-2. Furthermore, a significant decrease in the severity of EAE (P < 0.05), and an improvement in motor function (P < 0.05) and coordination (P < 0.05) were observed after treatment with a high dose of CuB. Our results suggest that CuB may have a wide-ranging effect on autoimmune responses in MS via a reduction in STAT3 activation, microgliosis, and adaptation of the IL-23/IL-17 axis. Further studies are needed to investigate the exact effect of CuB in glial cells and its efficiency and bioavailability in other neuroinflammatory diseases.

Preparation, characterization and pharmacokinetics of Cucurbitacin B solid dispersion

Cucurbitacin B (CuB) is a type of tetracyclic triterpenoids derived from the Cucurbitaceae melon, which has various biological activities. However, the poor water solubility and low bioavailability of CuB limit its wide range of clinical applications. ObjectiveThe present study aimed at the preparation of CuB to increase its solubility and further improve the oral bioavailability by using solid dispersion system (SD). MethodsThe optimal carrier was screened by crystallization inhibition and solubility test, and CuB-SD was further prepared by the solvent method. The optimal formulation was screened using dissolution as an indicator and characterized by techniques like DSC, XRPD, FTIR and SEM. The pharmacokinetic study was conducted to explore the potential of CuB-SD for oral administration. ResultsFrom the dissolution study, the cumulative dissolution of CuB prepared by the solvent method was nearly 6 times higher than the CuB. Moreover, DSC, XRPD and SEM analyses indicated that partial transformation of CuB into an amorphous form in SD formulations. FTIR demonstrated that there was hydrogen bonding between CuB and the carrier, which supported the higher solubility and dissolution of the CuB-SD. The in vivo pharmacokinetic study, UPLC-MS/MS was used to measure the concentration of CuB in plasma. In vivo study indicated that the formation of CuB-SD could largely improve the absorption of CuB, the AUC0-24h of CuB-SD (1:7) formulation was 3.6-fold higher than pure CuB. The AUC0-∞ of CuB-SD (1:7) was 692.44 ± 33.24 (ng/mL)*h, which was 498.77 ± 26.27 (ng/mL)*h and 187.41 ± 10.41 (ng/mL)*h for CuB-SD (1:5) and CuB, respectively. ConclusionsCollectively, CuB-SD was a promising way to enhance the oral bioavailability of CuB.

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 B, Purified and Characterized From the Rhizome of Corallocarpus epigaeus Exhibits Anti-Melanoma Potential.

The ethnomedicinal plant from the Cucurbitaceae family, Corallocarpus epigaeus, or its bioactive derivatives have been widely utilized in traditional medicine owing to their distinct applications against various human ailments and have lured the interest of ethnobotanists and biochemists. Here, we report for the first time, the anti-cancer potential of a bio-active fraction isolated from the dried rhizome of C. epigaeus, and the bioactive principle identified as cucurbitacin B (Cu-B). The purification processes involving the utilization of multiple organic extracts of C. epigaeus rhizome powder, yielded Cu-B from the Ethyl acetate Cytotoxic Fraction (ECF), obtained by the chromatographic separation of the ethyl acetate extract. Amongst the various cancer lines tested, melanoma cells exhibit maximal sensitivity towards the Cu-B-containing ECF fraction. Cu-B induces an apoptotic mode of cell death initiated intrinsically as well as extrinsically in A375 melanoma cells whilst remaining comparatively less toxic to normal skin fibroblasts. In vivo studies involving a NOD-SCID murine model of human melanoma demonstrate the ability of Cu-B to attenuate tumor growth, while being pharmacologically safe in vivo, as assessed in Swiss albino mice. Furthermore, Cu-B inhibits MEK 1/2 as well as the constitutive and EGF-induced ERK 1/2 activation, indicating a definitive involvement of MAPK signal transducers in regulating Cu-B-mediated anti-melanoma activity. Together, our study demonstrates the anti-melanoma potential of C. epigaeus-derived Cu-B, which indicates the Cucurbitaceae succulent as a prospective source for deriving potent and pharmacologically safe anti-cancer compounds.

Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78-FOXM1-KIF20A pathway.

Conjunctival melanoma (CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B (CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kd value of 0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78–FOXM1–KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.

Allosteric Regulation of IGF2BP1 as a Novel Strategy for the Activation of Tumor Immune Microenvironment.

Tumor immune microenvironment (TIME) regulators are promising cancer immunotherapeutic targets. IGF2BP1, as a crucial N6-methyladenosine (m6A) reader protein, recognizes m6A target transcripts, ultimately leading to cancer development. However, currently, the biological function of IGF2BP1 in regulating the TIME is not well-understood. In this study, we report that IGF2BP1 knockdown induces cancer cell apoptosis, thereby significantly not only activating immune cell infiltration including CD4+, CD8+ T cells, CD56+ NK cells, and F4/80+ macrophage but also decreasing PD-L1 expression in hepatocellular carcinoma (HCC). Then, chemical genetics identifies a small-molecule cucurbitacin B (CuB), which directly targets IGF2BP1 at a unique site (Cys253) in the KH1–2 domains. This leads to a pharmacological allosteric effect to block IGF2BP1 recognition of m6A mRNA targets such as c-MYC, which is highly associated with cell apoptosis and immune response. In vivo, CuB exhibits an obvious anti-HCC effect through inducing apoptosis and subsequently recruits immune cells to tumor microenvironment as well as blocking PD-L1 expression. Collectively, IGF2BP1 may serve as a novel pharmacological allosteric target for anticancer therapeutics via mediating TIME.

Cucurbitacin B and Evodiamine imparts anti‐neoplastic effects against Glioblastoma Multiforme via PI3‐K/AKT signaling pathway

Glioblastoma Multiforme (GBM) is a highly malignant and aggressive brain tumor. Patients’ survival is around 6 to 12 months even with chemotherapy and surgery. Temozolomide is a first line chemotherapeutic treatment prescribed along with surgery but due to the high resistance to the drug and a low patient survival rate, new drugs and drug combinations must continue to be investigated and developed. Evodiamine, a marketed supplement, and Cucurbitacin B, a compound obtained from natural plants, have been identified as promising candidates to treat various neoplasms but has not been well studied in GBM, and especially in combination. The present study investigated the anti‐proliferating effects of Evodiamine (Evo) and Cucurbitacin‐B (CuB) individually and in combination in U‐87MG cell line (Human Primary Glioblastoma cell line) and elucidated probable mechanisms of action via PI3K/AKT pathway in GBM. Results demonstrated that both CuB and Evo individually decreased the U‐87MG cell viability in a dose dependent manner. The effect was pronounced with drug combination. CuB and Evo imparted their effect via the PI3K/P‐AKT signaling pathway. In addition, Cells treated with CuB and Evo showed a decrease in Bcl2, an anti‐apoptotic factor, and an increase in Bax, a pro‐apoptotic factor, expression; a hallmark of induction of apoptosis. The increased expression of Cleaved Caspase‐3 and p‐c Jun have also been observed. Overall, Cucurbitacin and evodiamine individually and especially in combination proved to be potential anti‐neoplastic agents in U‐87MG cells and may be considered as potential adjuvant therapies for GBM.

Simultaneous detection of anticancer compounds (Cucurbitacin I, B and E) and some pharmacological properties of Indian Blastania species

Cucurbitacins are the predominant tetracyclic tri-terpenoids found in Cucurbitaceae members and possess several biological properties including anticancer. Therefore, the study was aimed at extracting and estimating three anticancer compounds viz. cucurbitacin I (CUI), cucurbitacin B (CUB) and cucurbitacin E (CUE) from two Cucurbitaceae species [Blastania cerasiformis (Stocks) A. Meeuse and Blastania garcinii (Burm.f.) Cogn.]. For this, terpenoid fractions were successfully isolated from the leaves, unripened and ripened fruits of Blastania species and subjected to RP-HPLC analysis of cucurbitacins. The results highlighted the significant yield of CUI and CUB from unripened (1.86±0.02, 3.00±0.04 mg/g DW) and ripened fruits (2.46±0.02, 2.74±0.09 mg/g DW) of B. cerasiformis. Similarly, unripened fruits fraction of B. garcinii reported the presence of high CUI (2.05±0.02 mg/g DW), CUB (1.76±0.03 mg/g DW) and CUE (4.28±0.12 mg/g DW) content. LC-MS analysis confirmed the presence of CUB along with 18 other bioactive compounds from the studied Blastania species. The study was further extended to evaluate the total terpenoid content and pharmacological properties (antioxidant, antidiabetic, anticancer and antimicrobial) from the isolated terpenoid fractions. The highest terpenoid content was recorded from unripened fruits of B. cerasiformis (1.53±0.00 mg UAE/g DW) followed by B. garcinii (1.52±0.00 mg UAE/g DW). The maximum antioxidant potential was revealed from unripened fruits fractions of both Blastania species through DPPH (0.03±0.00 mg AAE/g DW), FRAP (0.06±0.00 mg Fe(II)/g) and phosphomolybdenum (0.34–0.41±0.00 mg AAE/g DW) assays. The employed antidiabetic assays marked the efficiency of leaves fractions for α-amylase inhibition (52.79±1.40, 47.87±0.63%) and unripened fruits fraction for α-glucosidase inhibition (94.71±0.53, 92.59±2.80%). The significant anticancer activity was recorded from ripened fruits of B. garcinii (IC50 45.04 µg/mL) and B. cerasiformis (IC50 156.00 µg/mL). In addition, isolated fractions exhibited maximum antimicrobial activity against Staphylococcus aureus (2–6 mm), Bacillus cereus (2–3 mm) and Bacillus subtilis (2–3.3 mm) when compared with other tested pathogens. The principal component analysis revealed the impact of high terpenoid content on significant antioxidant, antidiabetic and anticancer activities of fruits. Present investigation highlighted the medicinal potential of Blastania species against cancer, diabetes, oxidative stress and human pathogen related diseases.

Study on the chemotherapeutic effect and mechanism of cucurbitacin E on laryngeal cancer stem cells.

ObjectivesStudy on the chemotherapeutic effect and mechanism of cucurbitacin E (CuE) on laryngeal cancer stem cells.MethodsWe used flow cytometry to sort out CD133+ laryngeal cancer stem cells; trypan blue rejection assay to detect the survival rate of laryngeal cancer stem cells; Cell counting kit‐8 (CCK‐8) assay to detect the effect of CuE on the proliferation ability of stem cells and the chemotherapeutic potentiation of doxorubicin; Transwell assay to observe the effect of CuE on the migration ability of stem cells; and Western Blot to detect the effect of CuE on the expression level of stem cell‐associated proteins. The tumor volume of nude mice was measured at the end of the experiment, and paraffin sections of nude mice tumor tissues were prepared and stained with Hematoxylin and eosin (H&E). The expression of c‐MYC in tumor tissues of nude mice was further detected by immunohistochemistry, and the effect of CuE on the expression level of related proteins in tumor tissues of nude mice was detected by Western Blot.ResultsCuE reduced the survival rate, proliferation ability, and migration ability of laryngeal cancer stem cells in vitro, and that CuE had a chemotherapeutic potentiating effect on doxorubicin. The possible mechanism of the chemotherapeutic effect of CuE was to reduce the expression of c‐MYC protein, and the possible mechanism of chemotherapy synergy was to reduce the expression of ABCG2 and P‐gp protein.ConclusionCuE has a chemotherapeutic effect on laryngeal cancer stem cells, as well as a chemotherapy synergy.

Reactive Oxygen Species-Responsive Nanococktail With Self-Amplificated Drug Release for Efficient Co-Delivery of Paclitaxel/Cucurbitacin B and Synergistic Treatment of Gastric Cancer.

Application of drug combinations is a powerful strategy for the therapy of advanced gastric cancer. However, the clinical use of such combinations is greatly limited by the occurrence of severe systemic toxicity. Although polymeric-prodrug-based nanococktails can significantly reduce toxicity of drugs, they have been shown to have low intracellular drug release. To balance between efficacy and safety during application of polymeric-prodrug-based nanococktails, a reactive oxygen species (ROS)-responsive nanococktail (PCM) with self-amplification drug release was developed in this study. In summary, PCM micelles were co-assembled from ROS-sensitive cucurbitacin B (CuB) and paclitaxel (PTX) polymeric prodrug, which were fabricated by covalently grafting PTX and CuB to dextran via an ROS-sensitive linkage. To minimize the side effects of the PCM micelles, a polymeric-prodrug strategy was employed to prevent premature leakage. Once it entered cancer cells, PCM released CuB and PTX in response to ROS. Moreover, the released CuB further promoted ROS generation, which in turn enhanced drug release for better therapeutic effects. In vivo antitumor experiments showed that the PCM-treated group had lower tumor burden (tumor weight was reduced by 92%), but bodyweight loss was not significant. These results indicate that the developed polymeric prodrug, with a self-amplification drug release nanococktail strategy, can be an effective and safe strategy for cancer management.

Cucurbitacin E Triggers Cellular Senescence in Colon Cancer Cells via Regulating the miR-371b-5p/TFAP4 Signaling Pathway.

The induction of cellular senescence is considered as a potent strategy to suppress cancer progression. Cucurbitacin E (CE) belongs to the triterpenoids and has received substantial attention for its antineoplastic property. However, the function of CE on cellular senescence remained elusive. Herein, we revealed that CE significantly induced cellular senescence in colorectal cancer (CRC) cells. The CE effects on the cellular senescence in CRC cells were confirmed by observing the common features of the senescence, such as the enhanced activity of senescence-associated β-galactosidase, γ-H2AX positive staining, and upregulation of senescence-associated proteins including p53, p27, and p21. Moreover, CE exerted pro-senescent effects in CRC cells via attenuating the transcription factor activating enhancer-binding protein 4 (TFAP4) expression, and the ectopic expression of TFAP4 blocked the CE-induced senescence. Mechanistically, CE treatment caused a robust increase in miR-371b-5p, which markedly repressed TFAP4. In contrast, silencing of miR-371b-5p counteracted the percentages of CE-induced senescent cells from 37.49 ± 2.61 to 7.06 ± 0.91% in HCT-116 cells via derepressing TFAP4 to attenuate the expression of p53, p21, and p16. Altogether, these results demonstrated that dietary CE induces CRC cellular senescence via modulating the miR-371b-5p/TFAP4 axis and presents opportunities for potential therapeutic strategies against CRC.

Cucurbitacin B inhibits TGF-β1-induced epithelial–mesenchymal transition (EMT) in NSCLC through regulating ROS and PI3K/Akt/mTOR pathways

BackgroundLung cancer is the leading cause of cancer mortality worldwide, and most of the patients after treatment with EGF-TKIs develop drug resistance, which is closely correlated with EMT. Cucurbitacin B (CuB) is a natural product of the Chinese herb Cucurbitaceae plant, which has a favorable role in anti-inflammation and anti-cancer activities. However, the effect of CuB on EMT is still far from fully explored. In this study, the inhibition effect of CuB on EMT was investigated.MethodsIn this study, TGF-β1 was used to induce EMT in A549 cells. MTS assay was used to detect the cell viability of CuB co-treated with TGF-β1. Wound healing assay and transwell assay were used to determine the migration and invasion capacity of cells. Flow cytometry and fluorescence microscope were used to detect the ROS level in cells. Western blotting assay and immunofluorescence assay were used to detect the proteins expression. Gefitinib was used to establish EGF-TKI resistant NSCLC cells. B16-F10 intravenous injection mice model was used to evaluate the effect of CuB on lung cancer metastasis in vivo. Caliper IVIS Lumina and HE staining were used to detect the lung cancer metastasis of mice.ResultsIn this study, the results indicated that CuB inhibited TGF-β1-induced EMT in A549 cells through reversing the cell morphology changes of EMT, increasing the protein expression of E-cadherin, decreasing the proteins expression of N-cadherin and Vimentin, suppressing the migration and invasion ability. CuB also decreased the ROS production and p-PI3K, p-Akt and p-mTOR expression in TGF-β1-induced EMT in A549 cells. Furthermore, Gefitinib resistant A549 cells (A549-GR) were well established, which has the EMT characteristics, and CuB could inhibit the EMT in A549-GR cells through ROS and PI3K/Akt/mTOR pathways. In vivo study showed that CuB inhibited the lung cancer metastasis effectively through intratracheal administration.ConclusionCuB inhibits EMT in TGF-β1-induced A549 cells and Gefitinib resistant A549 cells through decreasing ROS production and PI3K/Akt/mTOR signaling pathway. In vivo study validated that CuB inhibits lung cancer metastasis in mice. The study may be supporting CuB as a promising therapeutic agent for NSCLC and Gefitinib resistant NSCLC.

Cucurbitacin B exerts neuroprotection in a murine Alzheimer's disease model by modulating oxidative stress, inflammation, and neurotransmitter levels.

Alzheimer's disease (AD) type dementia encompasses diverse cognitive deficits marked by free radicals and pro-inflammatory cytokines mediated progressive neurodegeneration and vascular damage including the blood-brain barrier. Subsequently, an imbalance in neurotransmitters, excitotoxicity, and synaptic loss provide impetus to AD pathogenesis and perpetuate brain dysfunctions. Cucurbitacin possesses several biological properties and has shown potential in cancer, diabetes, and brain disorders. In this study, neuroprotective effects of cucurbitacin B (CuB) were investigated using the intracerebroventricular streptozotocin (STZ-ICV) AD prototype. Wistar rats (adult males) were injected STZ-ICV (3 mg/kg) bilaterally on day(s) 1 and 3. Rats were treated with CuB (25, 50 mg/kg, i.p.) or donepezil (1 mg/kg, i.p.) for 28 days daily starting from day 1. Behavioral tests viz. locomotor activity, motor coordination, and memory functions were conducted at different time intervals. After behavioral tests, biochemical markers of oxidative mutilation, inflammatory cell demise, and neurotransmitters were assessed in the whole brain. CuB attenuated STZ-ICV-induced decrease in spatial memory in novel object recognition task and long-term memory in passive avoidance test. CuB diminished protein carbonyls, lipid peroxidation, 8-hydroxy-2'-deoxyguanosine, and enhanced antioxidants in the brain of rats inoculated with STZ-ICV. A decline in inflammatory and cell death biomarkers was observed in rats treated with CuB and STZ-ICV. In neurotransmitter analysis, a decrease in acetylcholinesterase activity and glutamate levels indicated an increase in cholinergic and attenuation of excitatory transmission in the brain. GABA (γ-aminobutyric acid) levels were enhanced by CuB treatment in the STZ-ICV rat model. Histomorphometry analysis disclosed that CuB treatment caused an increase in viable neuron density in the cortex and hippocampus of rats against STZ-ICV neurotoxicity. It can be inferred that CuB can afford a decline in AD symptoms. CuB protects neurons against STZ-ICV toxicity that improved memory functions in rats.