|Year : 2018 | Volume
| Issue : 9 | Page : 505-511
Anti-tumor effects of phenolic alkaloids of menispermum dauricum on gastric cancer in vivo and in vitro
Di Wu1, Jiankuo Du1, Yan Zhang2, Yunming Su2, Hongfeng Zhang1
1 Department of Gastrointestinal Surgery, The Affiliated Tumor Hospital of Harbin Medical University, Harbin 150040, Heilongjiang Province, China
2 Department of Pharmacology, School Basic Medical Sciences, Heilongjiang University of Chinese Medicine, Harbin 150040, Heilongjiang Province, China
|Date of Web Publication||29-Jun-2018|
Department of Gastrointestinal Surgery, The Affiliated Tumor Hospital of Harbin Medical University, Harbin 150040, Heilongjiang Province
Source of Support: None, Conflict of Interest: None
Aim: This study was conducted to investigate the anti-tumor effects of the Chinese traditional herb phenolic alkaloids of menispermum dauricum (PAMD) on gastric cancer both in vitro and in vivo.
Materials and Methods: Cell apoptosis was detected in cultured SGC-7901 cells after administration of a different dose of PAMD. Gastric cancer model was established by single i.p. injection of SGC-7901 cells in the mice (n = 60). Then, animals were received high dose (20 mg/kg), medial dose (10 mg/kg), and low dose (5 mg/kg) of PAMD. Mice received 5-floxuridine was set as positive controls and received normal saline was as blank controls. Effects of PAMD on tumor growth were evaluated by tumor inhibition rate. Tumor tissues were collected from mice and detected for the expression of several genes P53, B-cell CLL/lymphoma 2 (BCL-2), BCL-2-associated X protein (BAX), CASPASE-3, K-RAS by real-time polymerase chain reaction, and Western blot. In addition, tumor cell changes were observed under transmission electron microscopy.
Results: The apoptosis index in PAMD at high- and medial-dose group was significantly higher than that in blank control group (P < 0.01). PAMD at different dose could significantly decrease the tumor weight compared to the blank control group (P < 0.01). In addition, PAMD could obviously increase BAX and caspase-3 expression as well as decrease K-RAS expression when compared to the blank control treatment (P < 0.01). Furthermore, PAMD could induce tumor cell morphology changes.
Conclusions: PAMD could suppress gastric tumor growth in vivo, possibly through increasing the expression of pro-apoptotic genes expression then leading to cell apoptosis and inhibiting oncogenic K-RAS expression.
Keywords: Cell apoptosis, gastric cancer, in vivo, K-RAS, phenolic alkaloids of menispermum dauricum
|How to cite this article:|
Wu D, Du J, Zhang Y, Su Y, Zhang H. Anti-tumor effects of phenolic alkaloids of menispermum dauricum on gastric cancer in vivo and in vitro. J Can Res Ther 2018;14, Suppl S2:505-11
|How to cite this URL:|
Wu D, Du J, Zhang Y, Su Y, Zhang H. Anti-tumor effects of phenolic alkaloids of menispermum dauricum on gastric cancer in vivo and in vitro. J Can Res Ther [serial online] 2018 [cited 2019 Sep 16];14:505-11. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/505/184521
| > Introduction|| |
Gastric cancer is the fourth most commonly diagnosed cancer, which is also the second leading cause of cancer-related death worldwide. Although considerable improvements were achieved recently, the treatment of gastric cancer remains still extremely unsatisfactory. Surgery is considered the gold standard for the treatment of gastric cancer while approximately two-thirds of patients have high possibilities of recurrence and metastasis after surgeries. In addition, limited effects of conventional chemotherapy on inhibiting gastric cancer growth lead to few promising results of this treatment. Therefore, it is important to search and develop new and more effective drugs for the treatment of gastric cancer.
Recently, traditional Chinese medicine (TCM) displayed an increasingly important role in the prevention and treatment of tumors due to the advantage of proven safety. According to the previous report, at least 70% of all drugs approved by the Food and Drug Administration for cancer were extracted from traditional medicine or natural sources during the past 30 years., Menispermum dauricum rhizome is a natural product which is widely used in the treatment of cardiovascular and thrombosis disorders in China. Phenolic alkaloids of menispermum dauricum (PAMD) are a mixture of a fat-soluble alkaloid extracted from dried roots of menispermum dauricum, which are among the major pharmacologic constituents of this plant. The main active components have been demonstrated to be the dauricine and daurisoline. As previous studies presented, PAMD mixture showed some inhibitory effects on arrhythmia, myocardial ischemia, thrombosis, and hypertension and it also has been identified to have anti-inflammatory and bacteria-inhibiting effects.,,, In addition, the main active components of PAMD dauricine has been demonstrated to have much more biological activities, including protection of cerebral injury, induction of cell apoptosis, suppression of tumor growth, cell invasion, and angiogenesis as well as prevention of drug resistance in some tumor cells.,, However, few studies have been conducted on the anti-tumor effects of PAMD on gastric cancer.
Cell apoptosis is the process of programmed cell death, whose disturbance has been demonstrated to be involved in the process of tumor growth and development. The effects of PAMD on tumor apoptosis were few. P53, B-cell CLL/lymphoma 2 (BCL-2), BCL-2-associated X protein (BAX), and CASPASE-3 were all important apoptosis-associated genes, which play important roles in tumor cell apoptosis through different signaling pathways. In this study, we examined the effects of PAMD on tumor cell apoptosis through detection of the expression of these apoptosis-associated factors. Moreover, K-RAS has been known as an oncogene, whose mutations are found in many cancers, such as colon cancer, pancreatic cancer, and lung cancer.,, In this study, we also explored the effects of PAMD on gastric tumor through the detection of the expression of K-RAS.
Owing to the widely biological effects and potential inhibitory effects on a malignant tumor of PAMD, the main aim of this study was to investigate the anti-tumor effects of PAMD on gastric cancer both in vitro and in vivo. First, we examined the effects of PAMD on gastric tumor cell apoptosis using SGC-7901 cells, and then we explored the anti-tumor effects of PAMD through constructing a mouse gastric cancer model. In addition, we also explored the underlying mechanisms of PAMD inhibiting-tumor effects through detecting the expression of apoptosis-related genes and oncogene.
| > Materials and Methods|| |
PAMD powder which was provided by Prof Dong Wang from Heilongjiang University of TCM was initially dissolved in a certain amount of dimethyl sulfoxide (DMSO, Sigma Chemical, St. Louis, MO, USA) and then diluted in phosphate buffer saline solutions. After filtration sterilization in a 0.22 μm millipore filter, PAMD solutions with a concentration of 400 μg/ml was prepared and stored at 4°C for later use.
Human gastric cancer cell line SGC-7901 was purchased from Keygen Biology Co., Ltd., (Nanjing, Jiangsu, China) and cultured in RPMI 1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Sijiqing Bio-engineering Material Institute, Hangzhou, Zhejiang, China) in a humidified atmosphere of 5% CO2 in air and at 37°C.
Apoptosis in SGC-7901 cell
SGC-7901 cells were seeded at 1 × 105/ml with a volume of 2.5 ml per well in the 6-well plates. Twenty-four hours after incubation, cells were treated with PAMD at different concentrations (20 mg/L as high dose, 10 mg/L as medial dose, 5 mg/L as low dose). Furthermore, cells treated with 5-floxuridine (5-FU, Sigma Chemical, St. Louis, MO, USA) (20 mg/L) were defined as positive controls while cells treated with RPMI 1640 medium were defined as blank controls. After 24 h, cells in all groups were stained with Hoechest 33342 (Sigma-Aldrich, St Louis, MO, USA) at the concentration of 5 μg/ml for 10 min and then stained with propidium iodide (PI, Sigma-Aldrich, St Louis, MO, USA) (40 μg/ml) for 20 min at 4°C in the dark according to the manual provided by the manufacturer. The late apoptotic cells were defined as Hoechst 33342+/PI + cells as described previously, and the red fluorescence and the blue fluorescence were observed under the fluorescence microscope (Olympus, Tokyo, Japan). The apoptosis of SGC-7901 cells was determined by the apoptosis index (AI), which was calculated as number of apoptotic cells/number of total cells.
Nude mice, weighing 20 ± 2 g, half male, and half female were obtained from Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine. Animals were maintained in a specific pathogen-free condition in our laboratory. Animal Ethics Committee of Heilongjiang University of Chinese Medicine approved this study, and all protocols were performed according to ethical guidelines. A total of 60 nude mice were randomly divided into five groups: PAMD treatment groups (high-, medium-, and low-dose group), positive control group, and blank control group, and all of them were received single i.p. injection of 200 μL SGC-7901 cells (1 × 106). Seven days postinjection, mice in the high-, medium-, and low-dose of PAMD group were injected i.p. with 20 mg/kg, 10 mg/kg, 5 mg/kg PAMD every day, respectively, mice in positive control group were given 5-FU for 20 mg/kg once a week. In addition, mice in blank control group were given the same volume of normal saline. The treatment for mice lasted 3 weeks. On the next day after the last final administration, the primary stomach tumors were dissected and weighed at the sacrifice. The tumor inhibition rate was calculated according to the following formula:
Tumor inhibition rate (%) = (The average tumor weight in blank control group-the average tumor weight in different experimental group)/the average tumor weight in blank control group.
Real-time polymerase chain reaction
Total RNA was extracted from the dissected tumor tissues using the TRizol reagent (Invitrogen, Carlsbad, CA, USA) according to the provided protocol. Briefly, 1 ml TRizol reagent was added to 50 mg tumor tissues in a 1.5 ml microcentrifuge tube. Then, samples were vortex-mixed until all tissue debris dissolved. Afterward, the mixture was added with 0.2 ml chloroform to remove proteins. After centrifugation, the supernatant was transferred to a separate tube mixed with 0.5 ml isopropanol to precipitate the RNA. Next, RNA pellet was rinsed with 75% ethanol, air dried, and dissolved in proper amount diethyl phosphorocyanidate treated H2O. RNA quantity and quality were determined by spectrophotometry at 260 nm and electrophoresis by 1% agarose gel. The first-strand of cDNA was transcribed using a PrimeScript Reverse Transcription System (Takara, Shiga, Japan), β-actin was used as an internal control to normalize for differences in input RNA. Primers for several genes were shown in [Table 1]. The polymerase chain reaction procedures were as follows: 1 cycle at 95°C for 10 s, 40 cycles at 95°C for 5 s, and 60°C for 34 s. Amplification was analyzed using △△Ct method. All the experiments were repeated three times over multiple days.
|Table 1: The primes of the genes used for real-time polymerase chain reaction|
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Tumor samples of mice from different treatment groups were ground and then homogenized with lysis buffer (10 mM Tris-HCl, pH 7.4; 0.15 M NaCl; 5 mM ethylene diamine tetraacetic acid, pH 8.0; 1% Triton X-100; 5 mM, dithiothreitol; 0.1 mM phenylmethanesulfonyl fluoride, and 5 mM ε-aminocaproic acid). After centrifugation, total tumor protein was quantified using a standard bicinchoninic acid (BCA) assay (Harbin Saituo Biotechnology, Heilongjiang, China). First, bovine serum albumin (BSA) obtained from BCA assay kit was dissolved in Tris-buffered saline containing 0.2% Tween-20 (TBST). Then, different volumes (0, 1, 2, 4, 8, 12, 16, and 20 μl) of BSA solution (0.5 mg/ml BSA) were added into 96-well plates to generate a concentration gradient, which was defined as standard samples. Meanwhile, 20 μl of tumor samples were added into another wells in the same plates. Next, 200 μl of working solution obtained from BCA assay kit was added into each wells followed by incubation at 37°C for 30 min. After that, chemiluminescence reaction was detected when optical density at 562 nm. Total tumor protein was quantified after the establishment of standard curve linear regression equation based on standard concentration and their corresponding optical density (OD) values.
For each sample, 50 μg protein was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), and blocked in 5% of milk in 0.1% TBST at room temperature for 1.5 h. After washing with TBST for three times, the membranes were incubated with mouse anti-K-RAS monoclonal antibody (Santa Cruz Biotechnology Inc., California, USA) and mouse anti-β-actin monoclonal antibody (Xiangsheng Biotechnology Inc., Shanghai, China), respectively. Both of them were diluted at 1:200 and incubated overnight at 4°C. The membranes were then washed with TBST and incubated with horseradish peroxidase–conjugated rabbit anti-mouse secondary antibodies (Harbin Saituo Biotechnology Inc., Heilongjiang, China). The immunoreactive bands were visualized by chemiluminescence and detected by an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK). All the experiments were repeated three times over multiple days.
Transmission electron microscopy
The marginal tumor tissues were detached and fixed in 2.5% glutaraldehyde at 4°C for 2 h. Then tumor mass was washed with 0.1 mol/L phosphate buffer. After that, the samples were fixed again in 1% osmic acid for 1.5 h and then dehydrated, penetrated, and embedded. Afterward, tumor samples were sliced using ultra-thin slicing machine and dually stained with uranium acetate and lead citrate. Finally, the treated tissues were observed under transmission electron microscope (Olympus, Tokyo, Japan).
Data were presented as the mean ± standard deviation from at least three separate experiments, and significance was analyzed using one-way analysis of variance followed by Student–Newman–Keuls multiple range test. A P < 0.05 was considered statistically significant. All the analyses were carried out with the SPSS 19.0 (SPSS Inc., Chicago, IL, USA).
| > Results|| |
Phenolic alkaloids of menispermum dauricum inhibits gastric tumor growth in a mouse model
Following the results in our study [Figure 1], compared to the blank control group, significant decrease of tumor weight was observed in mice after treatment of PAMD at high-, medial-, low-dose (P < 0.01), the tumor inhibition rates in the groups of PAMD high-, medial-, low-dose were 34.76%, 32.38%, and 31.00%, respectively. There was no significant difference among three PAMD groups. However, tumor weight decreased significantly in the positive control group when compared to high-, medial-, or low-dose groups (P < 0.05), and the tumor inhibition rate was also higher in the positive control group of 53.16%.
|Figure 1: Effects of phenolic alkaloids of menispermum dauricum on tumor weight after different treatments. Data were presents as mean ± standard deviation, **indicates P < 0.01|
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Phenolic alkaloids of menispermum dauricum could induce cell apoptosis in vitro
[Figure 2]a showed the apoptotic cells in different groups (Hoechst 33342+/PI + cells). Following the statistical analysis in [Figure 2]b, when compared to the blank control group, there was a significant increase of AI in medial-dose PAMD group, high-dose PAMD group, and positive control group (P < 0.01), and the highest AI (43.0% (43.0%) was observed in high-dose PAMD group. After multiple comparisons, there was no significant difference of AI between high-dose PAMD group and positive control group (P > 0.05) while the AI in both groups were significantly higher than the medial-dose PAMD group (P < 0.05). The AI in low-dose PAMD group was higher than the blank control group while there was not significant statistical difference.
|Figure 2: Phenolic alkaloids of menispermum dauricum inducing SGC-7901 cells apoptosis in vitro. (a) The fluorescence results stained with Hoechest33342 and propidium iodide; (b) Statistical results of apoptosis index. Data were presents as mean ± standard deviation, **indicates P < 0.01|
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Phenolic alkaloids of menispermum dauricum promotes the expression of apoptosis-related genes in the mRNA level
The results for the expression of several apoptosis-related genes including P53, BCL-2, BAX, and CASPASE-3 were shown in [Figure 3]. In the positive control group, 5-FU could significantly increase the expression of P53, BAX and caspase-3 while decrease BCL-2 expression when compared to the blank control group (P < 0.05). In PAMD groups, PAMD at different dose could significantly promote the expression of BAX and caspase-3 when compared to the blank control (P < 0.05) while with no obvious effects on the expression of P53 and BCL-2. In addition, compared to the positive group, the promoting effects on BAX and caspase-3 expression of PAMD at low-, medial-, or high-dose remarkably reduced (P < 0.05).
|Figure 3: Effects of phenolic alkaloids of menispermum dauricum on the expression of several apoptosis-related genes. Data were presents as mean ± standard deviation, *indicates P < 0.05, **indicates P < 0.01|
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Phenolic alkaloids of menispermum dauricum decreases the expression of oncogene K-RAS in vivo
Following the results in our study [Figure 4]a, the expression of K-RAS was significantly inhibited in PAMD treatment groups at mRNA level when compared to the blank control group (P < 0.05), and there was no significant difference of inhibiting effects between PAMD at low-, medial-, or high-level and 5-FU (P > 0.05). The results of western blot were shown in [Figure 4]b, which clearly presented the obviously decreased expression of K-RAS protein in PAMD treatment groups (high-, medial-, and low-dose groups) and the positive control group.
|Figure 4: Effects of phenolic alkaloids of menispermum dauricum on the expression of oncogene K-RAS. Phenolic alkaloids of menispermum dauricum. (a) Real-time polymerase chain reaction results. Data were presents as mean ± standard deviation, **indicates P < 0.01. (b) Western blot results|
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Phenolic alkaloids of menispermum dauricum could induce detrimental changes of tumor cell morphology
Following the results of transmission electron microscopy [Figure 5], the cell mitochondria vacuoles degenerated, the heterochromatin agglutinated slightly in the PAMD high-dose group; in the medial-dose group, the intracellular organelles decreased significantly, nuclear swell, and the heterochromatin agglutinated in the blocks; in the low-dose group, the tumor cells occurred with the liquefied lesions; in the positive control group, the tumor cells narrowed significantly, nucleolus was rare, the volume decreased and the chromatin structure showed turbidity while in the blank control group, the mitochondrial in some tumor cells became larger, some of which swell, and the membrane was integral.
|Figure 5: Effects of phenolic alkaloids of menispermum dauricum on tumor cell morphology using transmission electron microscopy. Compared to that in the blank group, autophagic vacuoles and degenerated mitochondria were observed in high-, medial-, low-dose phenolic alkaloids of menispermum dauricum treatment groups and positive control group. The arrow indicated the autophagic vacuoles in different groups|
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| > Discussion|| |
As previous described, PAMD is a kind of Chinese herbs that have been widely used as a remedy for the treatment of some inflammatory diseases such as arrhythmia, myocardial ischemia, thrombosis, and hypertension. The main active component of PAMD dauricine has been demonstrated to be involved in anti-tumor effects on several tumors through inducing apoptosis, inhibiting proliferation and invasion. Only a few studies were conducted to demonstrate the anti-tumor effects of PAMD on tumors such as inhibiting the proliferation of human cancer cells, suppressing the tumor-related genes expression while lots of them were limited to publication in China. Therefore, we conducted the present study to systemically explore the anti-tumor effects of PAMD on tumors, especially for gastric cancer. Following the results in our study, PAMD could not only induce gastric cell apoptosis in vitro, but also regulate tumor cell apoptosis-related genes (BAX and Caspase-3) and oncogene K-RAS expression. In addition, PAMD showed a directly inhibitory effect on tumor growth in mice.
As described previously, the disturbance of cell apoptosis is involved in the process of tumor growth and development. Dauricine, which is of the main component of PAMD, had been reported to induce apoptosis. Jin et al. have reported that dauricine may induce apoptosis in cultured human bronchial epithelial cells and in lungs of CD-1 mice by targeting CYP3A. Yang et al. have demonstrated that dauricine could induce apoptosis by targeting nuclear factor-kappa B signaling pathway in colon cancer cells. In addition, Su et al. demonstrated that PAMD could suppress the proliferation of human tumor cells. In this study, we identified that PAMD could induce cell apoptosis in SGC-7901 cells. Not only that but we also demonstrated that the effects of PAMD on cell apoptosis might be mediated by increasing the expression of BAX and Caspase-3. BAX, a member of proapoptotic protein in BCL-2 family involving in the mitochondrial pathways, together with BCL-2 an anti-apoptotic protein is the best characterized apoptosis-related protein that is considered as the primary regulators of apoptosis., PAMD seemly did not influence the expression of BCL-2 in our study, but directly promoted the expression of effector molecules BAX, leading to mitochondrial outer-membrane permeabilization which is responsible for cytochrome c release. In addition, PAMD inducing apoptosis might be involved in the activation of caspase-3 that is another kind of primary regulators of apoptosis.
As described above, PAMD could induce cell apoptosis of mitochondrial pathways. We also observed the changes of tumor cell morphology, especially for the mitochondria under transmission electron microscopy. In normal tumor cells, the activity of mitochondria is extremely active while PAMD could induce mitochondria vacuoles degeneration, heterochromatin agglutination as well as nucleolus swelling, ultimately leading to cell death.
We also explored other anti-tumor effects of PAMD on gastric cancer, and PAMD could suppress the expression of K-RAS. K-RAS mutation is involved in many cancers.,, K-RAS has also been detected in patients with gastric cancer, which may be involved in the carcinogenesis of gastric cancer., In addition, K-RAS mutation in gastric cancer has been reported to be related to DNA mismatch repair deficiency. Therefore, the anti-tumor effects of PAMD might be executed by suppressing the abnormal expression of K-RAS in gastric cancer.
| > Conclusion|| |
The Chinese herb PAMD could inhibit tumor growth in the mice and its anti-tumor effects might be mediated by increasing the expression of proapoptotic genes BAX and caspase-3 and then inducing cell apoptosis as well as inhibiting the abnormal expression of oncogene K-RAS. Since the effects of PAMD on the expression of apoptosis-related genes were only detected in mRNA level, further study is still needed to confirm our results, especially exploring genes expression by protein assessment.
Financial support and sponsorship
This study was supported by Heilongjiang Postdoctoral Grant (NO: LBH-Z09007) and the Key Research of Science and Technology Plan Projects of Heilongjiang Province (NO: GC09C405-3).
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.
Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9-29.
Huo JG, Qin FX, Cai XT, Ju JM, Hu CP, Wang, ZG, et al.
Chinese medicine formula “Weikang Keli” induces autophagic cell death on human gastric cancer cell line SGC-7901. Phytomedicine 2013;20:159-65.
Sullivan RN, Findlay MP, Zalcberg J. Adjuvant and neoadjuvant therapy for gastric carcinoma. Am J Cancer 2006;5:111-21.
Collins I, Workman P. New approaches to molecular cancer therapeutics. Nat Chem Biol 2006;2:689-700.
Cragg GM, Grothaus PG, Newman DJ. Impact of natural products on developing new anti-cancer agents. Chem Rev 2009;109:3012-43.
Zhao B, Chen Y, Sun X, Zhou M, Ding J, Zhan JJ, et al.
Phenolic alkaloids from Menispermum dauricum
rhizome protect against brain ischemia injury via regulation of GLT-1, EAAC1 and ROS generation. Molecules 2012;17:2725-37.
Qian JQ. Cardiovascular pharmacological effects of bisbenzylisoquinoline alkaloid derivatives. Acta Pharmacol Sin 2002;23:1086-92.
Kong XY, Gong PL. Effect of phenolic alkaloids of Menispermum dauricum
on thrombosis and platelet aggregation. Yao Xue Xue Bao 2005;40:916-9.
Liu JG, Li R, Liu GQ. l-S.R-daurisoline protects cultured hippocampal neurons against glutamate neurotoxicity by reducing nitric oxide production. Zhongguo Yao Li Xue Bao 1999;20:21-6.
Su YM, Zhang C, Xiao JY, Gang HL, Wang ZG, Hua D. Effects of pamd on the proliferation human tumour cells of pc-3 and bt5637. Haerbin Yi Ke Da Xue Xue Bao 2007;2:14.
Wang J, Li Y, Zu XB, Chen MF, Qi L. Dauricine can inhibit the activity of proliferation of urinary tract tumor cells. Asian Pac J Trop Med 2012;5:973-6.
Yang ZF, Li CH, Wang X, Zhai CY, Yi ZF, Wang L, et al.
Dauricine induces apoptosis, inhibits proliferation and invasion through inhibiting NF-kappaB signaling pathway in colon cancer cells. J Cell Physiol 2010;225:266-75.
He L, Liu GQ. Interaction of multidrug resistance reversal agents with P-glycoprotein ATPase activity on blood-brain barrier. Acta Pharmacol Sin 2002;23:423-9.
Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature 2001;411:342-8.
Cotter TG. Apoptosis and cancer: The genesis of a research field. Nat Rev Cancer 2009;9:501-7.
Karapetis CS, Khambata-Ford S, Jonker DJ, O'Callaghan CJ, Tu D, Tebbutt NC, et al.
K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008;359:1757-65.
Pellegata NS, Sessa F, Renault B, Bonato M, Leone BE, Solcia E, et al.
K-ras and p53 gene mutations in pancreatic cancer: Ductal and nonductal tumors progress through different genetic lesions. Cancer Res 1994;54:1556-60.
Johnson L, Mercer K, Greenbaum D, Bronson RT, Crowley D, Tuveson DA, et al.
Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 2001;410:1111-6.
Zhang Y, Su H, Zhang M, Liu ST. The effects of phenolic alkaloids from Menisperum daurine
(PAMD) on fas of gastic cancer cell sgc-7901. J Mudanjiang Med Univ 2014;35:10-1.
Cui C, Cui NS, Wang P, Song SL, Liang H, Ji AG. Sulfated polysaccharide isolated from the sea cucumber Stichopus japonicus
against PC12 hypoxia/reoxygenation injury by inhibition of the MAPK signaling pathway. Cell Mol Neurobiol 2015;35:1081-92.
Rödel C, Grabenbauer GG, Papadopoulos T, Bigalke M, Günther K, Schick C, et al.
Apoptosis as a cellular predictor for histopathologic response to neoadjuvant radiochemotherapy in patients with rectal cancer. Int J Radiat Oncol Biol Phys 2002;52:294-303.
Su YM, Li YH, Liu K, Zhang Y, Cui Y, Li WJ. The influence of PMAD on the content of MMP-2 in BXPC-3 tumor tissues. J Chengdu Med Coll 2010;5:113-5.
Chen NH, Liu JW, Zhong JJ. Ganoderic acid T inhibits tumor invasion in vitro
and in vivo
through inhibition of MMP expression. Pharmacol Rep 2010;62:150-63.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C(T)) Method. Methods 2001;25:402-8.
Jin H, Shen S, Chen X, Zhong D, Zheng J. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice. Toxicol Appl Pharmacol 2012;261:248-54.
Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R, et al.
Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002;111:331-42.
Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, et al.
The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 2000;6:1389-99.
Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, et al.
tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 2000;14:2060-71.
Sun Y, Lin Y, Li H, Liu J, Sheng XH, Zhang WC. 2,5-hexanedione induces human ovarian granulosa cell apoptosis through BCL-2, BAX, and CASPASE-3 signaling pathways. Arch Toxicol 2012;86:205-15.
Hiyama T, Haruma K, Kitadai Y, Masuda H, Miyamoto M, Tanaka S, et al.
K-ras mutation in Helicobacter pylori
-associated chronic gastritis in patients with and without gastric cancer. Int J Cancer 2002;97:562-6.
Okumura T, Ericksen RE, Takaishi S, Wang SS, Dubeykovskiy Z, Shibata W, et al.
K-ras mutation targeted to gastric tissue progenitor cells results in chronic inflammation, an altered microenvironment, and progression to intraepithelial neoplasia. Cancer Res 2010;70:8435-45.
van Grieken NC, Aoyama T, Chambers PA, Bottomley D, Ward LC, Inam I, et al.
KRAS and BRAF mutations are rare and related to DNA mismatch repair deficiency in gastric cancer from the east and the west: Results from a large international multicentre study. Br J Cancer 2013;108:1495-501.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]