Effect Of Cucurbitacin Research Articles

Reduction of Cancer Stem Cells and Invasiveness of Human Melanoma and Breast Cancer by Cucurbitacin B from Lagenaria siceraria

Cucurbitacins are secondary metabolites that are commonly found in the Cucurbitacae family. Many biological properties have been reported for cucurbitacins, including anti-inflammatory, antioxidant, antiviral, anti-malaria, and anticancer properties. While studies for the anticancer property of cucurbitacins focus mostly on the cell-cycle progression and apoptosis, no study has considered the effect of cucurbitacin on other cancer behaviors. Here, we report cell-proliferation-based drug testing on random herbal extracts leading to the identification of cucurbitacin B as an anticancer compound. Interestingly, cucurbitacin B had no effect on the proliferation of rat embryonic myoblast cells. We also found that cucurbitacin B significantly reduced the invasiveness of at least two highly metastatic breast cancer and melanoma cells. Using known cancer stem-cell markers, we observed a significant reduction of the melanoma stem cells. Molecularly, cucurbitacin B caused reduction of the metastasis-promoting gene Snail in melanoma and one of the cancer stem cell markers, ALDH1A1 (aldehyde dehydrogenase 1 A1), in breast cancer. Finally, we report the potential toxicity of cucurbitacin B in developing skin tissue and the olfactory organ using zebrafish embryo. In summary, our study suggests the potential use of cucurbitacin B for cancer metastasis and relapse treatment.

Effect of cucurbitacin on malignant biological behavior of breast cancer cells, and its possible underlying mechanism

Purpose: To study the influence of cucurbitacin on malignant biological behavior of mammary carcinoma cells, and the likely mechanism involved.Methods: Human mammary carcinoma cell line MDA-MB-436 was selected for cell culture and treated with different concentrations of cucurbitacin. The effect of cucurbitacin on cell activity, cell colonyformation capacity, cell invasion, migration potential, matrix metalloproteinase-9 (MMP-9) activity, and levels of vascular endothelial growth factor A (VEGFA), epithelial calcium adhesion (E-cadherin), and neurogenic calcium adhesion (N-cadherin) were measured. Moreover, levels of wave protein (vimentin), phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated signaling transduction, and transcription activation factor 3 (p-STAT3) and phosphorylated protein kinase B (p-Akt) were determined.Results: With increase in cucurbitacin dose, there was significant decrease in cell viability, cell colony ratio, cell invasion and migration capacity, and expression levels of MMP-9, VEGFA, e-cadherin, ncadherin, vimentin, P-EGFR, P-STAT3 and p-Akt (p < 0.05).Conclusion: Cucurbitacin inhibits the proliferation, invasion, and migration of breast cancer cells by down-regulating the expressions of EGFR/STAT3/Akt signaling-related proteins, and inhibiting epithelial-mesenchymal transition transformation.

Anti-metastatic Potential of Natural Triterpenoid Cucurbitacin B Against Cholangiocarcinoma Cells by Targeting Src Protein.

Owing to the crucial role of Src in cancer metastasis, interruption of Src and its signaling has been considered a promising strategy for cancer metastasis treatment. Cucurbitacin B, a dietary triterpenoid, has been shown to possess anti-proliferative and apoptosis-inducing activities in cholangiocarcinoma (CCA) cells via suppressing the activation of FAK which is a main downstream Src effector. We hypothesized that cucurbitacin B might act as a Src suppressant which conferring anti-metastasis effect against CCA cells. To investigate this, the role of Src in regulating metastasis behavior of CCA cells and the effect of cucurbitacin B on Src-mediated metastatic phenotype of these cells were determined. The results showed that activation of Src significantly enhanced the migratory and invasive abilities of CCA cells. Molecular analysis revealed that Src-facilitated metastasis behavior of CCA cells occurred by modifying expression of a wide range of metastasis-related genes in the cells. Consistent with gene expression results, activation of Src significantly induced the protein expression of 2 important metastasis-associated molecules, MMP-9 and VEGF. Cucurbitacin B markedly suppressed activation of Src and its key effector, FAK. As a consequence, the alteration of expression profiles of metastasis-associated genes induced by Src activator in CCA cells was diminished by cucurbitacin B treatment. The compound also down-regulated Src-induced expression of MMP-9 and VEGF proteins in the cells. Moreover, molecular docking analysis revealed that cucurbitacin B could interact with Src kinase domain and possibly restrain the kinase from being activated by hindering the ATP binding. In conclusion, cucurbitacin B exhibited anti-metastatic property in CCA cells via negatively influencing Src and Src-related oncogenic signaling. This compound may therefore be a potential therapeutic drug for further development as an anti-Src agent for treatment of metastatic CCA.

Cucurbitacin B Activates Bitter-Sensing Gustatory Receptor Neurons via Gustatory Receptor 33a in Drosophila melanogaster.

The Gustatory system enables animals to detect toxic bitter chemicals, which is critical for insects to survive food induced toxicity. Cucurbitacin is widely present in plants such as cucumber and gourds that acts as an anti-herbivore chemical and an insecticide. Cucurbitacin has a harmful effect on insect larvae as well. Although various beneficial effects of cucurbitacin such as alleviating hyperglycemia have also been documented, it is not clear what kinds of molecular sensors are required to detect cucurbitacin in nature. Cucurbitacin B, a major bitter component of bitter melon, was applied to induce action potentials from sensilla of a mouth part of the fly, labellum. Here we identify that only Gr33a is required for activating bitter-sensing gustatory receptor neurons by cucurbitacin B among available 26 Grs, 23 Irs, 11 Trp mutants, and 26 Gr-RNAi lines. We further investigated the difference between control and Gr33a mutant by analyzing binary food choice assay. We also measured toxic effect of Cucurbitacin B over 0.01 mM range. Our findings uncover the molecular sensor of cucurbitacin B in Drosophila melanogaster. We propose that the discarded shell of Cucurbitaceae can be developed to make a new insecticide.

The effects of cucurbitacin E on GADD45β-trigger G2/M arrest and JNK-independent pathway in brain cancer cells.

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti‐proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma‐astrocytoma U‐87‐MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin‐B1 disassociation were investigated by quantitative real‐time PCR and Western blot analysis. Based on our results, CuE showed growth‐inhibiting effects on GBM 8401 and U‐87‐MG cells. Moreover, GADD45β caused the accumulation of CuE‐treated G2/M‐phase cells. The disassociation of the CDC2/cyclin‐B1 complex demonstrated the known effects of CuE against GBM 8401 and U‐87‐MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.

Anticancer activity of 23,24-dihydrocucurbitacin B against the HeLa human cervical cell line is due to apoptosis and G2/M cell cycle arrest.

Cervical cancer is one of the primary causes of cancer-associated mortality worldwide. Due to the increasing incidence of cervical cancer, multiple treatment options are required. Initial responses to chemotherapy and surgical interventions are generally positive, however patients often experience relapse and tumor recurrence. Currently, the effects of cucurbitacins on different types of cancer are being investigated, as they exhibit a wide variety of bioactivities. The anticancer activity of the cucurbitacin 23,24-dihydrocucurbitacin B against a panel of human cervical cancer cell lines was investigated in the current study. Cell viability was determined using an MTT assay and apoptosis was detected using DAPI staining. The proportion of apoptotic cells, cell cycle distribution, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were estimated using flow cytometry. Protein expression was determined using western blot analysis. The results of the current study indicated that 23,24-dihydrocucurbitacin B inhibited the viability of human cervical cancer cell lines and had an IC50 of 40-60 µM. However, its cytotoxic effects were much less pronounced in normal epithelial fr2 and HerEpiC cells, where it had an IC50 of 125 µM. The underlying mechanisms of this were further studied and the results demonstrated that 23,24-dihydrocucurbitacin B induced apoptosis in HeLa cells and caused ROS-mediated shifts in the ΔΨm. Additionally, it caused the cell cycle arrest of HeLa cells at the G2/M checkpoint. The phosphoinositide 3 kinase/protein kinase B/mechanistic target of rampamycin (PI3K/AKT/mTOR) cascade may serve an important role in cancer tumorigenesis, progression and resistance to chemotherapy. The results indicated that 23,24-dihydrocucurbitacin B significantly decreased the expression of important proteins in the PI3K/Akt/mTOR cascade. Taken together, these results suggest that 23,24-dihydrocucurbitacin B may be novel method of treating cervical cancer.

Abstract 3224: Cucurbitacin D inhibits prostate tumor growth via targeting glucose metabolism

Abstract Background: Emergence of hormone-refractory PrCa (HRPC) after the anti-androgen therapy, cancer metastasis and chemo-resistance are the major hurdle for the treatment of prostate cancer (PrCa) patients. Accumulative evidence suggests that altered glucose metabolism is one of the mechanisms for metastatic PrCa cell survival and chemo-resistance. Therefore, identification and generation of natural/or synthetic pharmacological agents that can limit altered glucose metabolism might be highly useful for the treatment of metastatic and chemo-resistant PrCa. Cucurbitacins have shown potent anti-cancer and glucagonostic activities along with severe liver toxicity. Thus, a focus on developing different analogues of cucurbitacin is being pursued by scientific community. Herein, we report that Cucurbitacin D, an analogue of cucurbitacin, suppresses the growth of metastatic PrCa cells in vitro and in vivo via targeting glucose metabolism and its associated molecular targets. Methods: HRPC cells (PC3 and DU145) was used as a model system. Effect of Cucurbitacin D on PrCa cell proliferation and apoptosis was performed by MTS, xCELLigence and Annexin V assays. Effect of Cucurbitacin D on clonogenic potential of PrCa cells was examined by colony formation assay. In silico analysis was performed to study if Cucurbitacin D interacts with GLUT1 receptor. Effect of Cucurbitacin D on glucose metabolism of PrCa cells was performed by metabolic shift, glucose and lactate uptake assays. Effect of Cucurbitacin D treatment on key molecules of cell survival and glucose metabolism signaling pathways in PrCa cells was analyzed by Western blot and qRT-PCR analyses. Therapeutic efficacy of Cucurbitacin D against PrCa was evaluated in an ectopic PrCa xenograft mouse model. Results: Cucurbitacin D (0.1 to 1 µM) treatment significantly (P<0.01) inhibited the growth and metastatic potential of PrCa cells in a dose-dependent manner. Cucurbitacin D effectively induced apoptosis in PrCa cells as shown by enhanced Annexin V staining and PARP protein cleavage and arrested cells in G2/M phase via modulation of cell cycle regulatory proteins (inhibition of cyclin D1/E and Mcl-1 and induction of p21 and p27). Cucurbitacin D treatment dose-dependently decreased the lactate production, glucose uptake in PrCa cells which was correlated by suppressed expression of GLUT1 and its proficient docking with GLUT1 (with binding energy for this complex is -8.5 kcal mol-1). It has been reported that miR-132 targets the GLUT1, interestingly, Cucurbitacin D replenished the expression of miR-132 in PrCa cells. These growth inhibitory effects of Cucurbitacin D were confirmed in PrCa xenograft mouse model while administered intra-tumorally (1 mg/kg body weight thrice/week). Conclusion: Our results suggest that Cucurbitacin D is a novel modulator of glucose metabolism which could be a promising therapeutic modality alone or in combination of conventional chemotherapeutic agents for PrCa. Citation Format: Mohammed Sikander, Bilal Bin Hafeez, Shabnam Malik, Aditya Ganju, Fathi T. Halaweish, Murali Mohan Yallapu, Subhash C. Chauhan, Meena Jaggi. Cucurbitacin D inhibits prostate tumor growth via targeting glucose metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3224. doi:10.1158/1538-7445.AM2017-3224

Effects of cucurbitacin I on in vitro proliferation of HaCaT cells and the expression of keratin 17, signal transducer and activator of transcription 3 and vascular endothelial growth factor

Objective To evaluate effects of cucurbitacin I on in vitro proliferation of HaCaT cells and the expression of keratin 17 (K17) , signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) in cultured HaCaT cells. Methods In vitro cultured HaCaT cells were divided into 6 groups to be treated with cucurbitacin I at different concentrations of 0.0125, 0.025, 0.05 and 0.1 μmol/L (0.0125, 0.025, 0.05 and 0.1 μmol/L cucurbitacin I groups) , DMEM containing the same volume of DMSO as 0.1 μmol/L cucurbitacin I (DMSO group) , DMEM (negative control group) and 10 nmol/L calcipotriol (positive control group) , respectively. Cell counting kit-8 (CCK8) assay was performed to assess in vitro cellular proliferative activity after 12-, 24-, 36-hour treatment, reverse transcription (RT) -PCR to measure the mRNA expression of K17 and VEGF in HaCaT cells after 24-hour treatment, and Western blot analysis to determine the protein expression of K17, STAT3, phosphorylated-STAT3 (p-STAT3) and VEGF after 24-hour treatment. Statistical analysis was carried out by one-way analysis of variance (ANOVA) , repeated measures ANOVA, Student-Newman-Keuls (SNK) -q test and Pearson correlation analysis. Results The proliferative activity of HaCaT cells started to decrease after 12-hour treatment with cucurbitacin I at the concentration of 0.0125 μmol/L. When the concentration of cucurbitacin I increased up to 0.1 μmol/L, the cell proliferation rates were inhibited by 43.00% ± 2.11% and 48.98% ± 2.27% after 24-and 36-hour treatment respectively. Compared with the negative control group, cucurbitacin I at different concentrations all could inhibit in vitro proliferation of HaCaT cells (all P < 0.05) , and the inhibitory effects increased gradually with the increase of cucurbitacin I concentration and treatment duration. After 24-hour treatment, the mRNA expression of K17 and VEGF and the protein expression of K17, VEGF and P-STAT3 were significantly decreased along with the increase of concentration of cucurbitacin I (all P < 0.05) . Conclusion Cucurbitacin I can inhibit in vitro proliferation of HaCaT cells, and down-regulate the mRNA expression of K17 and VEGF and protein expression of K17, VEGF and P-STAT3. Key words: Cucurbitacins; Keratin-17; STAT3 transcription factor; Vascular endothelial growth factors; HaCaT cells

Cucurbitacins: elucidation of their interactions with the cytoskeleton.

Cucurbitacins, a class of toxic tetracyclic triterpenoids in Cucurbitaceae, modulate many molecular targets. Here we investigated the interactions of cucurbitacin B, E and I with cytoskeletal proteins such as microtubule and actin filaments. The effects of cucurbitacin B, E and I on microtubules and actin filaments were studied in living cells (Hela and U2OS) and in vitro using GFP markers, immunofluorescence staining and in vitro tubulin polymerization assay. Cucurbitacin B, E and I apparently affected microtubule structures in living cells and cucurbitacin E inhibited tubulin polymerization in vitro with IC50 value of 566.91 ± 113.5 µM. Cucurbitacin E did not affect the nucleation but inhibited the growth phase and steady state during microtubule assembly in vitro. In addition, cucurbitacin B, E and I all altered mitotic spindles and induced the cell cycle arrest at G2/M phase. Moreover, they all showed potent effects on actin cytoskeleton by affecting actin filaments through the depolymerization and aggregation. The interactions of cucubitacin B, E and I with microtubules and actin filaments present new insights into their modes of action.

Cucurbitacin E induces apoptosis of human prostate cancer cells via cofilin-1 and mTORC1.

Cucurbitacin E is an important member of the cucurbitacin family and exhibits inhibitory effects in various types of cancer. Cucurbitacin is a potential antineoplastic drug; however, its anticancer effect in human prostate cancer (PC) remains unknown. The aim of the present study was to determine whether the effect of cucurbitacin E on the cell viability and apoptosis of the human PC cell line, LNCaP, was mediated by cofilin-1- and mammalian target of rapamycin (mTOR). The results of the present study demonstrated that cucurbitacin E significantly exhibited cytotoxicity, suppressed cell viability (P<0.0001) and induced apoptosis (P=0.0082) in LNCaP cells. In addition, it was demonstrated that treatment with cucurbitacin E significantly induced cofilin-1 (P=0.0031), p-mTOR (P=0.0022), AMP-activated protein kinase (AMPK; P=0.0048), cellular tumor antigen p53 (p53; P=0.0018) and caspase-9 (P=0.0026) protein expression in LNCaP cells, suggesting that cucurbitacin E exerts its effects on LNCaP cells through cofilin-1, mTOR, AMPK, p53 and caspase-9 signaling. These results suggested that cucurbitacin E maybe used as a therapeutic agent in the treatment of human PC.

ANTITUMOR AND APOPTOTIC EFFECTS OF CUCURBITACIN A IN A-549 LUNG CARCINOMA CELLS IS MEDIATED VIA G2/M CELL CYCLE ARREST AND M-TOR/PI3K/AKT SIGNALLING PATHWAY.

Background:The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study.Materials and Methods:MTT assay and clonogenic assay were carried out to study effects of this compound on cell cytotoxicity and colony forming tendency in A-549 cells. Moreover, phase and fluorescence microscopic techniques were used to examine the effects on cell morphology and induction of apoptosis. The effects on cell cycle phase distribution were investigated by flow cytometry and effects on m-TOR/PI3K/Akt signalling proteins were assessed by western blot analysis.Results:Results showed that cucurbitacin A induced dose-dependent cytotoxic effects along with suppressing the colony forming tendency in these cells. Cucurbitacin A also induced morphological changes in these cells featuring chromatin condensation, cell shrinkage and apoptotic body formation. G2/M phase cell cycle collapse was also induced by Cucurbitacin A along with inhibition of expression levels of m-TOR/PI3K/Akt proteins.Conclusions:In conclusion, cucurbitacin A inhibits cancer growth in A-549 NSCLC cells by inducing apoptosis, targeting m-TOR/PI3K/Akt signalling pathway and G2/M cell cycle.

Abstract 3763: Cucurbitacin B targets EGFR-mutant lung cancer cells via activation of sestrin-3

Abstract The standard therapy for advanced non-small cell lung cancer (NSCLC) is based on the presence of epidermal growth factor receptor (EGFR) mutations with a clinical response to the EGFR tyrosine kinase inhibitors. However, despite the initial efficacy of the treatments, almost all patients acquire drug resistance and develop relapse after variable periods of time. One limiting factor for the use of natural and dietary agents is that they exert their effect at high concentrations which are not physiologically attainable. Cucurbitacins are highly oxidized tetracyclic triterpenoids which are arbitrarily divided into twelve categories and are widely distributed in the plant kingdom. Cucurbitacin B is one of the most common and has the highest content in many plants. We found striking effect of cucurbitacin B (0.2-0.6 μM; 24h) on EGFR-mutant lung cancer cells. There was remarkable decrease in the viability of H1975 and H1650 EGFR-mutant lung cancer cells. However, only modest effect of cucurbitacin B occurred on the viability of normal human bronchial epithelial cells. Treatment with cucurbitacin B also resulted in the inhibition of EGFR-mutant H1650 lung cancer cells colonies in a dose-dependent manner with almost complete inhibition at two weeks of treatment. Besides other signaling pathways, over-expression of phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) is frequently observed in tumors with EGFR mutations. Therefore, inhibition of the PI3K/AKT/mTOR signaling represents a favorable approach for the treatment of lung cancer patients with EGFR mutations. Treatment with cucurbitacin B caused inhibition of PI3K/Akt/mTOR and STAT-3 signaling alongwith simultaneous activation of AMPKα levels in EGFR-mutant lung cancer cells. Sestrins are highly conserved gene family found in all multicellular organisms of the animal kingdom. It is known that there is one sestrin gene in invertebrates and three genes in vertebrates (sestrins 1, 2 and 3). The inhibition of mTOR by sestrins has an impact on many mTOR-reliant processes such as translation, cell growth and proliferation. We found that treatment with cucurbitacin B caused specific increase in the protein and mRNA expression of sestrin-3 in EGFR-mutant lung cancer cells. Mechanistically, cucurbitacin B targeted mTOR via activating cell intrinsic inhibitor sestrin-3. We found that cucurbitacin B specifically activates sestrin-3, which may have resulted in dramatic mTOR inhibition. These results were further confirmed by using sestrin-3 knockdown in cucurbitacin B-treated cells. Sestrin-3 knockdown significantly reduced the effect of cucurbitacin B on cell-viability in EGFR-mutant lung cancer cells. These findings suggest novel mechanism for the action of cucurbitacin B and suggest that it could be explored further as potential therapy for EGFR-mutant lung cancer. Citation Format: Naghma Khan, Farah Jajeh, Mohammad Imran Khan, Eiman Mukhtar, Hasan Mukhtar. Cucurbitacin B targets EGFR-mutant lung cancer cells via activation of sestrin-3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3763.

Cucurbitacin B inhibits proliferation, induces G2/M cycle arrest and autophagy without affecting apoptosis but enhances MTT reduction in PC12 cells

&lt;p class="Abstract"&gt;In the present study, the effect of cucurbitacin B (a natural product with anti-cancer effect) was studied on PC12 cells. It significantly reduced the cell number, changed cell morphology and inhibited colony formation while MTT results showed increased cell viability. Cucurbitacin B treatment increased activity of succinode hydrogenase. No alteration in the integrity of mem-brane, the release of lactic dehydrogenase, the mitochondrial membrane potential, and the expression of apoptotic proteins suggested that cucurbitacin B did not induce apoptosis. The cell cycle was remarkably arrested at G2/M phase. Furthermore, cucurbitacin B induced autophagy as evidence by accumulation of autophagic vacuoles and the increase of LC3II. In addition, cucurbitacin B up-regulated the expression of p-beclin-1, p-ULK1, p-Wee1, p21 and down-regulated p-mTOR, p-p70S6K, CDC25C, CDK1, Cyclin B1. In conclusion, cucurbitacin B inhibited PC12 proliferation but caused MTT pitfall. Cucurbitacin B induced G2/M cell cycle arrest, autophagy, but not the apoptosis in PC12 cells.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Enhanced antitumor activity of cucurbitacin B combined with cerulenin in osteosarcoma

&lt;p class="Abstract"&gt;In the present study, the effect of cucurbitacin B and cerulenin combination on osteosarcoma was investigated to develop an effective treatment regimen. The IC&lt;sub&gt;50 &lt;/sub&gt;values of cerulenin and cucurbitacin B combination in the proportion of 1:1, 1:2 and 2:1 were 3.5, 7.2 and 8.6 μg/mL, respectively. The combination index (CI) values of &amp;lt;0.95 for inhibition of growth and &amp;lt;0.93 for inhibition of SENP5 expression clearly indicated synergism between the two. The Q-value for combination of 1 μg/mL cerulenin and 1 μg/mL cucurbitacin B was 1.2. AI calculated for tumor tissues treated with a combination of cucurbitacin B and cerulenin (26.2 ± 8.4%) was higher than that of the tissues treated separately with cucurbitacin B (14.5 ± 6.8%) or cerulenin (12.6 ± 7.5%). The AI for untreated tumor tissues was 4.2 ± 1.5%. Thus, the combination of cucurbitacin B and cerulenin can be a promising regimen in the treatment of osteosarcoma.&lt;/p&gt;

Inhibitory effect of cucurbitacin B on proliferation of human colon cancer cells

Objective To investigate the inhibitory effect of cucurbitacin B on proliferation of human colon cancer cells.Methods LS174-T cells were treated with different concentrations of cucurbitacin B (0.01,0.10,1.00,10.00,and 100.00 mol/L) for 24,48 and 72 h.The cell proliferation was detected by cell counting kit-8 (CCK-8) method,and the distribution of cell cycle by flow cytometry.signaling transducers and activators of transcription 3 (STAT3),Caspase-3,and Tert mRNA were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR).Results CCK-8 results showed that after treatment with cucurbitacin B (0.01 and 100.00 moL/L) for 24 h,inhibitory rate of LS174-T cell proliferation was (5.22 ±2.02)％ and (73.90 ± 1.85)％ respectively.After treatment with 100.00 mol/L of cucurbitacin B for 24 h and 48 h,inhibitory rate was (73.90 ± 1.85)％ and (98.84 ±0.51)％ respectively.The statistically significant difference was dose-and time-dependent (P ＜ 0.05).Flow cytometry revealed that as compared with control group,the percentage of cells in G2/M phase in 1.00 μmol/L concentration group was increased [(6.33 ± 0.74) ％ and (30.43 ± 4.09) ％],and that in G0/G1 phase decreased [(78.5 ± 4.72) ％ and (43.2 ± 5.65) ％] with the difference being statistically significant (P ＜0.05).The FQ-PCR indicated that after treatment with cucurbitacin B of 0.01 and 10.00 mol/L for 24 h,STAT3 level was 0.750 0 ±0.003 8,0.660 0 ±0.005 5,respectively.After treatment with cucurbitacin B of 0.10 μmol/L for 24 h and 48 h,Caspase-3 mRNA level was 4.30 ±0.29 and 6.79 ±0.13 respectively with the difference being statistically significant (P ＜ 0.05).Conclusion Cucurbitacin B can inhibit proliferation of colon cancer cells. Key words: Cucurbitacin B; Colon cancer; Proliferation

Abstract 4109: Cucurbitacin B: A novel natural agent for the management of non-small cell lung cancer

Abstract Lung cancer is the primary cause of cancer death in both men and women in the United States and worldwide. The general prognosis is still very low despite of developments in the treatment due to improved surgical techniques, increased application of combined modality treatments and the use of new drugs. Since the past decade, researchers have been investigating a range of purified compounds from dietary sources as possible anticancer drugs. The main issue with the use of natural products is their effectiveness at high doses which generally could not be delivered to humans through dietary consumption. Here, we observed that cucurbitacin B exhibits antiproliferative effects on human non-small cell lung cancer (NSCLC) cells at very low concentrations. The cucurbitacins are highly diverse and oxygenated tetracyclic triterpenoids isolated from plants of Cucurbitaceae family which are well-known for their bitterness of edible products like pumpkins, gourds and squashes. Cucurbitacin B is one of the most abundant and has been most widely used. In this study, we determined the effect of cucurbitacin B on the inhibition of cell-growth and suppression of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling in human NSCLC cells. Treatment of cucurbitacin B (0.2-0.6 μM; 24 h) was found to result in 34-61% and 45-65% decrease in the viability of human NSCLC H1792 and A549 cells respectively, but had only minimal effect on normal human bronchial epithelial cells. The IC50 of cucurbitacin B in these cells ranged from 0.43-0.49 μM. The A549 cell colonies were also reduced by treatment with cucurbitacin B in a dose-dependent manner. It has been well documented that mTOR functions downstream of the PI3K/Akt pathway and is phosphorylated in response to stimuli that activate the PI3K/Akt pathway. Treatment of A549 cells with cucurbitacin B (0.2-0.6 μM; 24 h) caused decrease in the protein expression of PI3K (p85 and p110) and inhibition of the phosphorylation of Akt and mTOR. PI3K/Akt-dependent phosphorylation signals through tuberin, the protein product of tuberous sclerosis complex (TSC)1/TSC2 complex, which leads to activation of mTOR. The mTOR also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) which disassociates from the eukaryotic translation initiation factor (eIF) 4E, allowing activation of protein translation. Treatment of A549 cells with cucurbitacin B also caused inhibition of the phosphorylation of p70S6K1, eIF-4E and 4E-BP1 and downstream targets of mTOR. In cucurbitacin B treated cells, there was also inhibition of the constituents of mTOR signaling complex such as Rictor, Raptor, GβL and PRAS40 and activation of AMP-activated protein kinase (AMPKα) and tuberous sclerosis complex (TSC)2. We suggest that cucurbitacin B could be developed as an agent for the management of lung cancer. Citation Format: Naghma Khan, Farah Jajeh, Sameh M. Shabana, Hasan Mukhtar. Cucurbitacin B: A novel natural agent for the management of non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4109. doi:10.1158/1538-7445.AM2014-4109

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)-1β 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-1β 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.

Cucurbitacin E Induces G2/M Phase Arrest through STAT3/p53/p21 Signaling and Provokes ApoptosisviaFas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells

Cucurbitacin E, a tetracyclic triterpenes compound extracted from cucurbitaceous plants, has been shown to exhibit anticancer and anti-inflammatory activities. The purpose of this study was to elucidate whether cucurbitacin E promotes cell cycle arrest and induces apoptosis in T24 cells and further to explore the underlying molecular mechanisms. The effects of cucurbitacin E on T24 cell's growth and accompanied morphological changes were examined by MTT assay and a phase-contrast microscope. DNA content, mitochondrial membrane potential (ΔΨm) and annexin V/PI staining were determined by flow cytometry. The protein levels were measured by Western blotting. Our results demonstrated that cucurbitacin E-induced G2/M arrest was associated with a marked increase in the levels of p53, p21 and a decrease in phospho-signal transducer and activator of transcription 3 (STAT3), cyclin-dependent kinase 1 (CDK1) and cyclin B. Cucurbitacin E-triggered apoptosis was accompanied with up-regulation of Fas/CD95, truncated BID (t-BID) and a loss of ΔΨm, resulting in the releases of cytochrome c, apoptotic protease activating factor 1 (Apaf-1) and apoptosis-inducing factor (AIF), and sequential activation of caspase-8, caspase-9, and caspase-3. Our findings provided the first evidence that STAT3/p53/p21 signaling, Fas/CD95 and mitochondria-dependent pathways play critical roles in cucurbitacin E-induced G2/M phase arrest and apoptosis of T24 cells.

Effects of cucurbitacins on cell morphology are associated with sensitization of renal carcinoma cells to TRAIL-induced apoptosis.

Cucurbitacins B and D were among the compounds identified as sensitizers of cancer cells to TRAIL-mediated apoptosis in a high-throughput screen. Therefore a series of cucurbitacins was further investigated for TRAIL sensitization and possible mechanisms of action. A total of six cucurbitacins promoted TRAIL-induced apoptosis (B, I, E, C, D, and K) and one (P) was inactive. Sensitization of renal adenocarcinoma cells to TRAIL was apparent after as little as 1-4h pretreatment and did not require continued presence of cucurbitacin. Active cucurbitacins induced caspase-8 activation only after subsequent TRAIL addition and caspase activation was required for apoptosis suggesting amplified proximal signaling from TRAIL death receptors. Cucurbitacin-sensitized TRAIL-induced cytotoxicity was inhibited by N-acetyl cysteine. Structure-activity relationship analysis in comparison to published studies suggests that TRAIL-sensitizing and general cytotoxic activities of cucurbitacins may be decoupled. Cucurbitacins are reported to be inhibitors of STAT3 activation. However, their TRAIL-sensitizing activity is STAT3-independent. Treatment of renal carcinoma cells with active cucurbitacins produced rapid and dramatic changes in cell morphology and cytoskeletal organization (also prevented by NAC). Therefore, cucurbitacins may be useful as tools for investigating the molecular mechanism(s) of action of TRAIL sensitizers, particularly with regard to temporal aspects of sensitization and modulation of TRAIL signaling by cell morphology, and could form the basis for future therapeutic development in combination with TRAIL death receptor agonists.

Abstract 4235: Antiproliferative effects of cucurbitacin B in breast cancer cells: Down-regulate c-Myc/hTERT/telomerase pathway and obstruct the cell cycle

Abstract Abstract: Naturally occurring cucurbitacins have been shown to have anticancer, antimicrobial and anti-inflammatory activities. In this study, we determined the effects of cucurbitacin B extracted from Thai herb Trichosanthes cucumerina L. on the regulation of telomerase in three human breast cancer cell lines (T47D, SKBR-3, and MCF-7) and a mammary epithelium cell line (HBL-100). Cell viability after treatment with cucurbitacin B which is an active ingredient of this herb, was assessed by MTT assay and cell cycle analysis by flow cytometry. Telomeric Repeat Amplification Protocol (TRAP) assays and RT-PCR (qualitative and realtime) were performed to investigate activity of telomerase as well as expression of human telomerase reverse transcriptase (hTERT) and c-Myc. The c-Myc protein level was also determined in SKBR-3 and HBL-100. Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and an obvious inhibitory effect was seen in the estrogen receptor (ER)-negative breast cancer cell SKBR-3 rather than ER-positive MCF-7 and T47D cells. The expressions of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, the reduction of c-Myc protein after the treatment was clearly observed in SKBR-3 cells even at the ten times lower concentration of cucurbitacin B compared to the concentration used for the HBL-100. DNA flow cytometric analysis indicated that treatment with cucurbitacin B resulted in G2/M arrest. These results imply that cucurbitacin B exerts anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression and arrest of the cell cycle at G2/M phase in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2011-4235