Huachansu Capsule inhibits the proliferation of human gastric cancer cells via Akt/mTOR pathway.

BACKGROUND: For decades, the traditional Chinese medicine preparation, Huachansu Capsule (HCS), has been applied to a variety of solid tumors and leukemias with significant curative effects. More importantly, HCS has few side effects on cardiovascular and gastrointestinal functions in patients. However, the potential mechanism of the anti-tumor activity of HCS has not been fully revealed. The current study investigated the in vivo and in vitro effects of HCS on the proliferation and apoptosis of human gastric cancer (GC) cells and explored the underlying mechanism. MATERIALS AND METHODS: HCS was first diluted to varying concentrations followed by the treatment to MGC-803 and BGC-823 GC cells. Cell proliferation was evaluated by Cell Counting Kit-8 assay. Cell invasion and migration were assessed using Transwell membrane chambers. Apoptosis and cell cycle arrest in GC cells induced by HCS were detected by flow cytometry. Western blotting assays were used to measure the influence of HCS on apoptosis-related proteins, including B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Cleaved-Caspase-3. Additionally, mammalian target of rapamycin (mTOR) signaling pathway-related proteins such as phosphorylated (p)-Akt, p-mTOR and p-4E-BP1 were detected. Transmission electron microscopy was used to observe the microstructure of apoptotic cells. An animal imaging technique was used to analyze the influence of HCS on the growth of GC cells in vivo and immunohistochemistry assays were performed to investigate the signal transduction pathways influenced by HCS. RESULTS: HCS significantly inhibited the proliferation, invasion and migration of MGC-803 and BGC-823 GC cells. It also induced cell cycle arrest at the G2/M phase and increased the cell apoptotic rate. Additionally, the HCS treatment downregulated the protein levels of Bcl-2, but upregulated the protein expression of Bax and cleaved-caspase 3. Furthermore, HCS downregulated the levels of p-Akt, p-mTOR and p-4E-BP1, suggesting that HCS inhibited tumor growth of GC via suppressing the Akt/mTOR pathway. CONCLUSION: This study indicated that HCS has significant anti-proliferative and apoptotic effects both in vitro and in vivo, and that HCS can inhibit tumor growth of GC via suppressing the Akt/mTOR pathway and induce apoptosis through the intrinsic pathway. Our study provides a scientific basis for the clinical application of HCS.

Bufalin induces protective autophagy by Cbl-b regulating mTOR and ERK signaling pathways in gastric cancer cells.

Bufalin, a natural small-molecule compound derived from the traditional Chinese medicine Chan su, has shown promising anti-cancer effects against a broad variety of cancer cells through different mechanisms. It has been reported to induce autophagy in gastric cancer cells. However, the molecular mechanism involved is not fully elucidated. In the present study, we aimed to investigate the molecular mechanism by which bufalin induce autophagy in human gastric cancer cells. We found that bufalin induced apoptosis and autophagy in gastric cancer cells, and autophagy prevented human gastric cancer cells from undergoing apoptosis. Bufalin treatment changed the expression of autophagy-related proteins. Moreover, phosphorylated Akt, mTOR, and p70S6K were all significantly decreased, while phosphorylated ERK1/2 was increased by bufalin. Pretreatment of MGC803 cells with the ERK1/2-specific inhibitor PD98059 led to the down-regulation of LC3 II. Further study showed that Cbl-b positively regulated autophagy by suppressing mTOR and enhancing ERK1/2 activation. Therefore, our data provide evidence that bufalin induces autophagy in MGC803 cells via both Akt/mTOR/p70S6K and ERK signaling pathways, and Cbl-b-mediated suppression of mTOR and activation of ERK1/2 might play an important role.

Jinlong Capsule (JLC) inhibits proliferation and induces apoptosis in human gastric cancer cells in vivo and in vitro.

BACKGROUND: As a representative traditional Chinese medicine made by modern pharmaceutical technology, Jinlong Capsule (JLC) has been used for several decades to treat liver cancer with significantly improved clinical outcomes as adjuvant therapy. JLC consists of three medicinal animals including freshly prepared Bungarus, Agkistrodon and Gecko. The active components were extracted by the process of modern cryogenic and biochemical separation from raw animals. However, the specific molecular mechanisms underlying the antitumor activities of JLC were not fully investigated. In the current study, experiments were carried out to examine the effect of JLC on anti-proliferative, pro-apoptotic activities of human gastric cancer (GC) cell lines in vivo and in vitro. METHODS: MTT assay was used to observe the viability of MGC-803 and BGC-823 cells treated with JLC. Apoptosis and cell cycle distribution of MGC-803 and BGC-823 cells induced by JLC were analyzed by flow cytometry. Western blot assay was used to detect the effect of JLC on apoptosis-related proteins, including Bax, Bcl-2, survivin and caspase-3. Transmission electron microscopy (TEM) was used to evaluate the microstructure of apoptotic GC cells. Tumor growth in vivo was monitored using live-imaging system. Immunohistochemical staining (IHC) was used to examine the expression of apoptosis-related proteins in tumor tissues. RESULTS: Our data indicated that JLC inhibited proliferation and induced apoptosis of MGC-803 and BGC-823 cells in a concentration-dependent manner. JLC significantly inhibited tumor growth in nude mice. Both in vivo and in vitro studies showed that JLC could downregulate the expression of Bcl-2 and survivin, whereas upregulate the levels of bax and caspase-3. JLC had significant antitumor effects in human GC through cell cycle arresting. Besides, JLC altered the microstructure of GC cells. CONCLUSION: These findings demonstrate that JLC can be considered as a promising candidate in GC treatment.

Apoptosis of human gastric carcinoma cells induced by Euphorbia esula latex.

AIM: To investigate the effect of Euphorbia esula (E. esula) extract in inhibiting proliferation and inducing apoptosis in SGC-7901 cells. METHODS: E. esula extract at different concentrations was used to inhibit proliferation and induce apoptosis of human gastric carcinoma SGC-7901 cells. Inhibition of proliferation was detected with thiazolyl blue assay, and apoptosis was detected with fluorescence microscopy, transmission electron microscopy, and flow cytometry. The mechanisms were studied by measurement of caspase-3 and caspase-8 activities and Bax and Bcl2 mRNA expression. RESULTS: The thiazolyl blue assay showed that SGC-7901 cell viability and proliferation were inhibited significantly by E. esula extract in a time- and concentration-dependent manner. Fluorescence microscopy revealed that the cell nuclei showed the characteristic changes of apoptosis, such as uneven staining and chromatin marginalization. Some key features of apoptosis were also observed under transmission electron microscopy, which included cellular shrinkage and the foaming or bubbling phenomenon. When the cells were analyzed by flow cytometry, a sub-G1 peak could be seen clearly. Spectrophotometric assay of caspase-3 and caspase-8 activities in the treated cells showed an approximately two-fold increase. Reverse transcription polymerase chain reaction showed that Bax mRNA expression was upregulated, while Bcl2 mRNA expression was downregulated. CONCLUSION: E. esula extract inhibited proliferation and induced apoptosis in SGC-7901 cells, in a caspase-dependent manner, involving upregulation of Bax and downregulation of Bcl2.

A flavonoid compound from Chrysosplenium nudicaule inhibits growth and induces apoptosis of the human stomach cancer cell line SGC-7901.

CONTEXT: Gastric cancer remains highly prevalent, but treatment options are limited. Natural products have proved to be a rich source of anticancer drugs. Chrysosplenium nudicaule Ledeb. (Saxifragaceae) is a perennial herb that grows in the highlands of China. It has been used as a traditional Chinese medicine to treat digestive diseases for hundreds of years. Recent studies revealed that this herb had anticancer activity, and the flavonoids were speculated to be the effective components. 6,7,3'-Trimethoxy-3,5,4'-trihydroxy flavone (TTF) and 5,4'-dihydroxy-3,6,3'trimethoxy-flavone-7-O-ß-d-glucoside (DTFG) are flavonoid compounds isolated from Chrysosplenium nudicaule. OBJECTIVE: This study examined the effect of TTF and DTFG on SGC-7901 human stomach cancer cell in vitro to determine the anticancer and induction of apoptosis properties of TTF. MATERIALS AND METHODS: The proliferation of cells treated with 32, 16, 8, 4, and 2 µg/mL of TTF or DTFG for 24, 48, and 72 h was assessed by the MTT assay. After being treated with TTF, the apoptosis of SGC-7901 cells was assessed by acridine orange staining, ultrastructure, electrophoresis of DNA fragmentation, and flow cytometry. RESULTS: Results indicated that TTF inhibited the growth of cancer cells with an IC50 value of 8.33 µg/mL after 72 h incubation. However, DTFG showed no inhibitory effect on the growth of the cancer cell. Further studies on TTF also confirmed that it was able to induce apoptosis of SGC-7901 cells at a concentration as low as 4 µg/mL. DISCUSSION AND CONCLUSION: The apoptotic effect of TTF makes it a promising candidate for future chemotherapeutic application in treating stomach cancer.

Naturally occurring phenethyl isothiocyanate-induced inhibition of gastric cancer cell growth by disruption of microtubules.

BACKGROUND AND AIM: Phenethyl isothiocyanate (PEITC) derives from vegetables commonly consumed by man and has been demonstrated as a promising chemopreventive agent against several types of cancer. However, the potential in preventing gastric cancer as well as the underlying mechanisms are to date not fully understood. The present study aimed at elucidating the cellular effects induced by PEITC in gastric cancer cells leading to apoptosis. METHODS: The human gastric cancer cell lines Kato-III and MKN74 were employed. Cell proliferation was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphology and migration were investigated through a contrast microscope. Cell cycle distribution was analyzed using flow cytometry of PI-stained cells. Microtubules were studied by confocal detection of Kato-III cells transfected to express GFP-tagged microtubules. Commercial kits were employed to study the effect of PEITC on apoptosis, caspase-3 activity, and glutathione content in MKN74 cells. RESULTS: Kato-III and MKN74 cells responded, with different sensitivity, dose- and time-dependently in inhibition of cell proliferation to PEITC treatment. Further, PEITC induced aberrated cell morphologies and inhibited migration of MKN74 cells. Kato-III cells treated with PEITC accumulated in G2 /M phase and displayed a loss of microtubuli with the subsequent formation of apoptotic bodies. Although weak responses, MKN74 cells also accumulated in G2 /M phase, became apoptotic, increased caspase-3 activity, and suffered a reduction of glutathione pool. CONCLUSION: Our findings demonstrate that PEITC induces disintegration of microtubules in human gastric cancer cells contributing to cell cycle arrest and ultimately apoptosis, contributing to an increased understanding of PEITC-induced inhibition of gastric cancer cell growth.

Antioxidant activity and ultrastructural changes in gastric cancer cell lines induced by Northeastern Thai edible folk plant extracts.

BACKGROUND: Phytochemical products have a critical role in the drug discovery process. This promising possibility, however, necessitates the need to confirm their scientific verification before use. Hence, this study aims to evaluate (1) the antioxidant activity, (2) cytotoxicity potential, and (3) the effect on ultrastructural alteration in gastric cancer cell lines through exposure to fractions of three local Northeastern Thai edible plants. METHODS: Plants, Syzygium gratum, Justicia gangetica and Limnocharis flava were extracted with ethyl acetate, and each crude extract analysed for their total phenolics content by Folin-Ciocalteu method. Their antioxidant activity was assessed using the ABTS system. The extracts were then assayed for cytotoxicity on two gastric cancer cell lines Kato-III and NUGC-4, and compared with Hs27 fibroblasts as a control using the MTT assay. The cell viability (%), IC50 values, as well as the ultrastructural alterations were evaluated after treatment with one way analysis of variance (ANOVA). RESULTS: The total phenolic values of the ethyl acetate extracts were well correlated with the antioxidant capacity, with extracted product of S. gratum displaying the highest level of antioxidant activity (a 10-fold greater response) over J. gangetica and L. flava respectively. Exposure of S. gratum and J. gangetica extracts to normal cell lines (Hs27) resulted in marginal cytotoxicity effects. However, through a dose-dependent assay S. gratum and J. gangetica extracts produced cytotoxicological effects in just over 75 percent of Kato-III and NUGC-4 cell lines. In addition, apoptotic characteristic was shown under TEM in both cancer cell lines with these two extracts, whereas characteristics of autophagy was found in cell lines after post exposure to extracts from L. flava. CONCLUSIONS: From these three plants, S. gratum had the highest contents of phenolic compounds and antioxidant capacity. All of them found to contain compound(s) with cytotoxicity in vitro on cancer cells but not on normal cell lines as resolved in tissue culture and ultrastructural analysis. This is the first report to show the effect on cellular alteration as apoptosis of an ethyl acetate extract of S. gratum and J. gangetica. Further studies are now focused on individual isolates and their function, prioritizing on S. gratum and J. gangetica for the development of novel therapeutics and combatants against cancer.

Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells.

Effects of magnetic fields (MFs) on cancer cells may depend on cell type and exposure conditions. Gene expression levels are different among cancer cells. However, the effect of MFs on cancer cells with different gene expressions is still unclear. In this study, the cancer cell lines BGC-823, MKN-45, MKN-28, A549, SPC-A1, and LOVO were exposed to a low-frequency MF. Specific parameters of MFs were determined. Furthermore, the potential of the MF to influence cancer cell growth with midkine (MK) expression was evaluated. Cell proliferation and cell cycle were detected using the CCK-8 assay and flow cytometry. Cell ultrastructure was observed by transmission electron microscopy. BGC-823 cells with over-expression of MK (BGC-MK cells) and stanniocalcin-1 were generated by plasmid construction and transfection. Results showed that exposure to a 0.4-T, 7.5 Hz MF inhibited the proliferation of BGC-823, MKN-28, A549, and LOVO cells, but not MKN-45 and SPC-A1 cells. Moreover, the inhibitory effect of the MF on BGC-MK cells was lower (12.3%) than that of BGC-823 cells (20.3%). Analysis of the cell cycle showed that exposure to the MF led to a significant increase in the S phase in BGC-823 cells, but not in BGC-MK cells. In addition, organelle morphology was modified in BGC-823 cells exposed to the MF. These results suggest that exposure to a 0.4-T, 7.5 Hz MF could inhibit tumor cell proliferation and disturb the cell cycle. The alteration of MK expression in cancer cells may be related to the inhibitory effect of the MF on these cells.

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3.

AIM OF THE STUDY: Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated. MATERIALS AND METHODS: Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunofluorescence method. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Fluorescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate. CONCLUSIONS: Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.

Inhibition of apoptosis facilitates necrosis induced by cisplatin in gastric cancer cells.

Although cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, the potential interconnections between these modes of cell death induced by the drug remain unknown. We studied this phenomenon in gastric cancer cell lines and identified one cell line (SGC-7901) that underwent apoptosis, and another cell line (BGC-823) that primarily underwent nonapoptotic cell death, in response to cisplatin. Apoptosis in cisplatin-treated SGC-7901 cells seemed to be caspase dependent and was mediated, at least in part, by the BH3-only protein, Noxa. This was evidenced by the rapid upregulation of Noxa and inhibition of apoptosis by small interfering RNA knockdown of Noxa. Nonapoptotic cell death induced by cisplatin in BGC-823 cells was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase independence, plasma membrane disruption, and intracellular vacuole formation, indicative of necrosis. Surprisingly, blockage of apoptosis induction by a general caspase inhibitor or by Noxa small interfering RNA in SGC-7901 failed to protect against cisplatin-induced cell death. Under such conditions, SGC-7901 cells displayed cellular features associated with necrosis. Cisplatin-induced apoptosis, thus, seems to precede necrosis when the apoptotic machinery is operative. When the apoptosis program is defective, necrotic cell death takes place as an alternative pathway leading to cell demise. Induction of different modes of cell death that are interrelated in the same cells by cisplatin has the potential to be exploited in formulating new adjuvant cancer therapies.

[Effect of Weikangfu granule on ultrastructure of gastric mucosa in patients of precancerosis with spleen deficiency syndrome].

OBJECTIVE: To observe the effect of Weikangfu Granule (WKFG) on the ultrastructure of gastric mucosa. METHODS: WKFG was used to treat 61 patients with gastric mucosa intestinal metaplasia or atypical proliferation, and histopathological and ultrastructural examination on gastric mucosa of antral region obtained through gastroscopy were conducted before and after treatment. RESULTS: After treatment, the histopathology and intestinal metaplasia subset of antral focal region, and the background lesion in ultrastructure of the mucosa in the nonfocal region were all improved in the 4 groups, The effects of Spleen-Qi Deficiency syndrome group and Spleen-Yang Deficiency group were superior to Ying-Deficiency group as well as Spleen-Deficiency and Qi-Stagnation group (P < 0.05, P < 0.01), and approached to those in the healthy control group. CONCLUSION: WKFG improves the quantitative balance of internal bioactive substances to maintain the homeostasis, promotes the ultrastructural normalization of mucosa so as to cure the clinical symptoms and reverse the intestinal metaplasia and atypical proliferation of mucosa.

[Induction of apoptosis by tea polyphenols in MGC cells].

In order to search for tumor cells apoptosis inducer, the apoptosis effects and mechanism of tea polyphenols were studied. Tea polyphenols is an active compounds purified from tea. The apoptosis effects of tea polyphenols were observed on the human gastric carcinoma cells (MGC) by MTT reduction test, DNA agarose gel electrophoresis and transmission electron microscopy (TEM) technique. Having been treated by tea polyphenols in 125 micrograms.ml-1 for 24 h, DNA extracted from MGC cells showed a typical internuclesosomal DNA degradation, i.e., DNA ladder and apoptotic vehicles were observed under TEM. The effects of tea polyphenols were shown to parallel with its cytotoxic activity in MGC cells. These results suggest that the mechanism of antitumor action of tea polyphenols is related to its apoptosis inducing activity.

Differentiation of human gastric adenocarcinoma cell line MGc80-3 induced by verbascoside.

Verbascoside is a natural antioxidant extracted from Pedicularis striata Pall (Jueyehesen). After being treated with 20 mumol/l verbascoside, the growth curve and mitotic index of human gastric adenocarcinoma MGc80-3 cells decreased remarkably, cell doubling time was delayed, the cellular growth inhibitory rate amounted to 53.2%, cell surface charge assayed by cell electrophoresis obviously changed, the electrophoresis rate dropped from 3.51 microns/s/v/cm to 2.74, i.e., the percent of retardation reached 28.4%. There was a 75% decrease of the tumorigenicity for the treated cells compared with the untreated cells inoculated subcutaneously in BALB/C nude mice. Scanning electron microscopy revealed that the microvilli on the surface of treated cells had been reduced obviously. It confirmed that verbascoside, similar to DMSO, could reverse MGc80-3 cells' malignant phenotypic characteristics and induced redifferentiation of MGc80-3 cells.

[Study on mitochondrial ultrastructure, trace elements and correlative factors of gastric mucosa in patients with spleen deficiency syndrome].

Eighty-eight gastropathic patients with Spleen deficiency syndrome by using transmission electron microscope (TEM), X-ray energy disperse analysis system (EDAX), histochemical staining and radioimmuno methods were examined. The authors found that the gastric mucosa cAMP, SOD level, the quantity of mitochondria and its crista, the ratio of diameter between ventricle and cavity of mitochondria and the content of Zn, Cu of mitochondria were reduced in the trend of healthy control group, Spleen Qi deficiency group, Spleen deficiency with Qi stagnation group; chronic superficial gastritis group, chronic atrophic gastritis group, gastric cancer group: complete small intestinal metaplasia group, incomplete small intestinal metaplasia group, complete colonic intestinal metaplasia group, incomplete colonic intestinal metaplasia group (P < 0.05-0.001). While the degeneration rate of mitochondria, the Cu/Zn ratio of mitochondria, the metaplasia rate of gastric, the rate of incomplete colonic intestinal metaplasia and the content of serum LPO were increased in the above turn. It is suggested that the comprehensive effect of the degeneration of mitochondria and the quantitative changes of its correlative factors is the physiopathologic base for inducing Spleen deficiency disease, gastric mucosa metaplasia and canceration. Much attention must be paid in clinic to the cancerization trend of gastric disease with Spleen deficiency syndrome.

Lysosomal exocytosis induced by hyperthermia: a new model of cancer cell death. II. Effect on peritoneal macrophages.

Recently we described the "macrophagic lysosomal exocytosis" (MLE) as a possible new mode of cancer cell death induced by hyperthermia (HT) [1]. In order to confirm this first report, HT was applied at the peritoneum level with perfusional procedure, after catheter insertion under local anesthesia. We evaluated the subcellular changes of peritoneal macrophages in human gastric tumor, before and after hyperthermic treatment at 42 degrees C for 90 minutes. Transmission electron microscopy showed that treatment produced the disappearance of the cytoplasmic granules, with a consequent extracellular scavenger action of the phagocytic cells, the proliferation of some organelles such as mitochondria, endoplasmic reticulum and Golgi complex that may be addressed to a subsequent restoration of the granular pool in the degranulated macrophages. This phenomenon can enhance the already documented effect of hyperthermic perfusional chemotherapy in peritoneal tumors.

Preliminary investigations of a correlation between electron energy loss and morphometric analyses on ultrathin cryosections from normal and neoplastic gastric tissues.

Electron energy loss spectroscopy (EELS) has been used to measure the ratios of C, N, O, P and Ca in ultrathin cryosections from normal and neoplastic gastric tissues. First results show a correlation between the EELS, and morphometric data in cells from these tissues. We have found that ultrathin, freeze-dried cryosections, with an average thickness of up to 75 nm, are stable enough for EELS-analysis in a 200 KV electron microscope with an adapted Gatan-EELS-Spectrometer.

Gastric carcinoma with psammomatous calcification: report of a case, with reference to calculogenesis.

A case of gastric adenocarcinoma with argyrophilic property and psammomatous calcification is reported. Histologically, the psammoma bodies are found most frequently within the glandular lumina. Electron microscopy, however, reveals that calcium first appears within the cytoplasm of tumor cells. Electron probe x-ray microanalysis demonstrates calcium and phosphorus in granular or floccular osmiophilic deposits found in intracytoplasmic electronlucent zones of tumor cells. By x-ray diffractometry, the calcium component is presumed to be hydroxyappatite (Ca5(PO4)3.(OH). The findings strongly support the view of the intracytoplasmic origin of psammomatous calcification. The tumor yielded a parathyroid hormone (PTH)-like substance, and a possible relationship between this substance and psammomatous calcification is spectulated.