|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 Nov 18];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.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]