Research Progress on Anti-tumor Effect and Mechanism of Cucurbitacins / 中国实验方剂学杂志

Cancer is a major global public health problem. Statistics from the national cancer center of China also show that cancer has become a major disease threatening human health with increasing morbidity and mortality. The occurrence and development mechanism of cancer is complex， involving multiple stages， multiple genes and multiple signaling pathways. Conventional chemoradiotherapy and emerging targeted therapy methods are the main methods in treatment of tumor. However， the quality of life of patients as well as the sustained and effective therapeutic effect are seriously affected due to the toxic side effects and drug resistance. Therefore， it is the global focus to find safe and effective anti-cancer drugs. The research and development and application of anti-cancer herbal medicines such as paclitaxel， vinblastine， podophyllin， ginsenoside and ginseng polysaccharide have brought new hope for the treatment of cancer. Cucurbitacine from Chinese medicine cucurbitaceae plantsare is a kind of highly oxidized tetracyclic triterpene compound with extensive pharmacological effects and complex mechanism. In the family of cucurbitacines， cucurbitin B， D， E and I have been studied most frequently on anticancer effect， and in a large number of studies， they have been found to play an important role in tumor diseases of the digestive system， respiratory system， reproductive system， blood system， urinary system and so on. With significant effect in inhibiting tumor cells proliferation， blocking the cell cycle， inducing apoptosis and autophagy death， inhibiting cell migration and invasion， inhibiting tumor angiogenesis， regulating the levels of reactive oxygen species and regulating immune system， such cucurbitacins are expected to be developed as a new kind of anti-cancer drugs. The authors of this study aim to provide reference for the further research and development of new anti-cancer drugs about cucurbitines by summarizing the anti-tumor effect and mechanism of the cucurbitacins.

Cucurbitacin B inhibits proliferation and invasion of osteosarcoma 143b cells and promotes apoptosis / 肿瘤

Objective: To investigate the effects of cucurbitacin B (CuB) on the proliferation, invasion and apoptosis of osteosarcoma 143B cells, to explore the related molecular mechanisms. Methods: 143B cells were treated with different concentrations of CuB. The proliferation ability of 143B cells was detected by crystal violet staining and colony formation assay. The invasion ability of 143B cells was detected by Transwell chamber assay. The apoptosis rate and cell cycle distribution were detected by flow cytometry (FCM) method. The expression levels of epithelial-mesenchymal transition (EMT)-related markers including Vimentin, Snail and matrix metalloproteinase-9 (MMP-9) mRNAs and proteins were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The expression levels of apoptosis-related proteins [Bad, cleaved-caspase 3, cleaved-polyadenosine diphosphate ribose polymerase (PARP) and cyclin B1] and protein kinase B (PKB, Akt) signal pathwayrelated proteins [Akt, phospho-Akt (p-Akt), phosphatase and tensin homolog deleted on chromosome ten (PTEN) and phospho-PTEN (p-PTEN)] were detected by Western blotting. Results: Compared with the untreated control group, the proliferation and invasion of 143B cells treated with different concentrations of CuB were significantly inhibited (all P < 0.05). After treatment with 30 or 40 nmol/L CuB, the apoptosis rate of 143B cells was increased (both P < 0.05), the cell cycle was blocked in G2/M phase (both P < 0.01). The expression levels of MMP-9, Vimentin and Snail mRNAs and proteins were remarkably down-regulated (all P < 0.05) in 143B cells treated with CuB. The expression levels of apoptosis-related Bad, cleavedcaspase 3 and cleaved-PARP proteins were remarkably up-regulated (all P < 0.05) in 143B cells treated with CuB, the expression of cell cycle-related cyclin B1 protein was down-regulated (all P < 0.01) in 143B cells treated with CuB. At the same time, CuB reduced the expression level of p-Akt protein and increased the expression level of p-PTEN protein (both P < 0.05). Conclusion: CuB can inhibit the proliferation and invasion of osteosarcoma 143B cells, and promote apoptosis. These effects may be related to Akt pathway impediment and EMT processing attenuation.

Mechanism of cucurbitacin B underlying pressure overload-induced myocardial fibrosis / 中华老年心脑血管病杂志

Objective To study the mechanism of cucurbitacin B (CuB) underlying pressure over load-induced myocardial fibrosis.Methods Sixty C57 mice were divided into sham operation group,CuB treatment group,aorta ligation group,CuB+aorta ligation group (15 in each group).The animals received intragastric gavage (0.2 mg/kg · 2 d) one week after operation and a myocardial fibrosis model of mice was induced by aorta ligation 4 weeks after operation.Microvascular density (MVD) was detected with immunohistochemical staining.Expressions of α-SMA,CD31,CD34,vimentin and endothelial-mesenchymal transition (EndMT) were detected by Western blot with immunofluorescence staining.Results No significant difference was found in cardiac MVD,and expression level of α-SMA,vimentin,CD31 and CD34 between sham operation group and CuB group 4 weeks after operation (P＞0.05).The cardiac MVD and expression level of CD31 and CD34 were significantly lower while the expression level of α-SMA and vimentin was significantly higher in aorta ligation group than in sham operation group 4 weeks after operation (P＜0.05).The cardiac MVD and expression level of CD31 and CD34 were significantly higher while the expression level of α-SMA and vimentin was significantly lower in CuB+aorta ligation group than in aorta ligation group 4 weeks after operation (P＜0.05).Conclusion CuB can attenuate cardiac fibrosis by regulating EndMT.

Low Nanomolar Cucurbitacin-I Induces G2 / M Phase Arrest and Apoptosis in Gastric Cancer Cells and the Underlying Mechanism / 胃肠病学

Background:Upon inhibition of STAT3 signaling pathway,cucurbitacin-I elicits anticancer effect in various malignancies. However,the anticancer effect and underlying mechanism of cucurbitacin-I in gastric cancer is still elusive. Aims:To explore the effect of low nanomolar cucurbitacin-I on cell proliferation,cell cycle and apoptosis in human gastric cancer cells and the underlying mechanism in vitro. Methods:Human gastric adenocarcinoma cell lines AGS and HGC-27 were treated with cucurbitacin-I at low nanomolar concentration. The anti-proliferative effect of cucurbitacin-I was detected by CCK-8 assay and colony formation assay. Flow cytometry was used to assess the cell cycle and apoptosis. Expressions of cell cycle-related proteins,as well as activation of related pathways such as caspase-3 / PARP apoptotic pathway,STAT3, GADD45α and JNK/ p38 MAPK signaling pathways were determined by Western blotting. Results:Cucurbitacin-I markedly inhibited the growth of gastric cancer cells at low nanomolar concentration by inducing G2 / M phase arrest and apoptosis via a STAT3-independent manner. Furthermore,it was revealed that the anticancer effect of cucurbitacin-I was associated with up-regulation of GADD45α,activation of JNK/ p38 MAPK signaling pathway and the subsequent apoptotic events. Conclusions:The present study provides new insights into the mechanism of anticancer effect of cucurbitacin-I, supporting cucurbitacin-I as an attractive therapeutic drug in gastric cancer.

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 / 中华皮肤科杂志

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 pmol/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.

Standardization of Ethanolic Extract of Cucurbita Maxima Seed.

Standardization is an important step for the establishment of a consistent biological activity, a consistent chemical profile, or simply a quality assurance program for production and manufacturing of herbal drugs preparation of any herbal formulation identification, evaluation and standardization is rudimentary identification involves the morphology, microscopy parameter of plants, evaluation and standardization of herbal drugs includes physical, chemical and biological parameters. These parameters are crucial for preparation of accurate and potent formulation. The present communication attempts to investigate pharmacognostical and phytochemical details of Cucurbita maxima, (Cucurbitaceae). The Preliminary phytochemical analysis revealed presence of carbohydrates, alkaloids, flavonoids, saponins, proteins and amino acids in alcoholic extract. HPTLC studies reveal that alcoholic extract gives 3 spots and alcoholic extract depicts 5 spots on the TLC plate in Butanol: acetic: water solvent system with Ninhydrin as spraying agent and 3 spots with vanillin as spraying agent and with butanol: phenol: water (6:1:1) 4 spots were seen with Ninhydrin as spraying agent and 2 spots were seen with vanillin as spraying agents. The GC/MS of pet ether methyl ester showed number of peaks. Out of which 3 highest peaks in descending order were taken into consideration. OSAZONE were formed which showed needle shaped crystals of glucaosazone. The study revealed specific identities for which may play a key role in identification of plant and can be useful in standardization of the herbal drugs.

Assessment of effects on health due to consumption of bitter bottle gourd (Lagenaria siceraria) juice.

Background & objectives: The bottle gourd (Lagenaria siceraria) is popularly known as lauki, ghia or dudhi in India. Its consumption is advocated by traditional healers for controlling diabetes mellitus, hypertension, liver diseases, weight loss and other associated benefits. However, in last few years there have been reports of suspected toxicity due to consumption of its juice. This led to the constitution of an Expert Committee by Department of Health Research at Indian Council of Medical Research (ICMR), Ministry of Health & Family Welfare, Government of India in October 2010. The committee looked into the issues related to safety of consumption of bottle gourd juice, and this paper presents the findings. Methods: Information on cases of suspected toxicity due to consumption of bottle gourd juice was collected by internet search, advertising on website of ICMR and by writing to State and district health authorities as well as to medical colleges, hospitals and private nursing homes across the country. Results: Three deaths were reported, one from Delhi and two from Uttar Pradesh after consumption of extremely bitter bottle gourd juice. Three persons who died after consumption of freshly prepared bottle gourd juice or juice mixed with bitter gourd (karela) juice were over 59 years of age and had diabetes since last 20 years. This juice was reported to be extremely bitter by all three. Twenty six persons were admitted to various hospitals of the country on complaint of abdominal pain and vomiting following consumption of freshly prepared bottle gourd juice. Diarrhoea and vomiting of blood (haematemesis) was reported in 18 (69.2%) and 19 (73.1%) patients, respectively. Biochemical investigations revealed elevated levels of liver enzymes. More than 50 per cent patients had hypotension. Endoscopic findings showed profusely bleeding stomach with excessive ulceration seen in distal oesophagus, stomach and duodenum in most of the cases. All these patients recovered fully and no sequeale was recorded for any of the cases. Interpretation & conclusions: Cucurbitaceae family, of which bottle gourd is a member contains the toxic tetracyclic triterpenoid compounds called cucurbitacins which are responsible for the bitter taste. There is no known antidote for this toxicity and clinicians treat such cases symptomatically only. The Committee made the following recommendations: (i) The community needs to be educated that bitter tasting bottle gourd juice should not be consumed and in case there is any discomfort, nausea, vomiting, diarrhoea or any feeling of uneasiness after consumption of juice, the person should immediately be taken to a nearby hospital. (ii) Clinicians are suggested that patients coming with symptoms (discomfort, nausea, vomiting, diarrhoea, gastrointestinal bleeding after consumption of juice) should immediately be attended to and general supportive care should be provided, i.e. IV fluids/crystalloids/blood products/ fresh frozen plasma to maintain the haemodynamics and electrolyte balance; Ryle’s tube to be put in for gastric lavage and to assess gastrointestinal (GI) bleed- aspirate to be preserved; Proton pump inhibitors should be given for management of GI bleed and appropriate treatment for other complications should be given. (iii) The possible research areas identified are chemical composition studies on bitter and normal bottle gourd and other members of cucurbitaceae family; animal toxicity studies and studies on interaction between bottle gourd juice and other drugs.