Molecule action mechanisms of NM-3 on human gastric cancer SGC-7901 cells in vivo or in vitro

Abstract

AIM: To study the molecule action mechanisms of NM-3 on the growth of human gastric cancer SGC-7901 cells in vivo or in vitro.

METHODS: SGC-7901 from human non-differentiated gastric cancer cell line was cultured with NM-3 at 100 mg/mL for 24 h. We observed its inhibitory rate and the density of micro-vascular growth in grafted mice with human gastric cancer SGC-7901. The apoptosis of human gastric cancer SGC-7901 was revealed in NM-3 treatment group by using terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate-fluorescene nick end labeling (TUNEL) method and flow cytometry analysis.

RESULTS: The growth of SGC-7901 cells was markedly inhibited compared with control growp, which was smaller than that in normal saline control group (4.17 g ± 0.22 g vs 9.45 g ± 1.38 g, P < 0.01). The level of apoptosis of human gastric cell line SGC-7901 was obviously increased in NM-3 treatment group at 1 mg.L^-1 for 24 h. NM-3 inducing apoptotic index in NM-3 plus carboplatin group was 3.5 times that of carboplatin control group (TUNEL: 27.98% ± 6.12% vs 12.94% ± 2.12%, FACScan: 26.86% ± 5.69% vs 11.86% ± 1.09%, P < 0.01). Western blot analysis showed that the apoptotic index of human gastric cancer was elevated for 12, 24 and 36 h with an evident time-effect relationship in groups at 100 mg.L^-1. NM-3 enhanced the inhibitive effects and sensitivity of chemotherapy for human gastric cancer in nude mice. These results suggested that NM-3 played a key inhibitive role in the growth of grafted human gastric cancer in nude mice.

CONCLUSION: NM-3 can inhibit the growth of human gastric cancer cell line SGC-7901, and enhance the sensitivity of carboplatin on SGC-7901 and induced its apoptosis.

INTRODUCTION

Apoptosis plays a key role in the proliferation and turnover of malignant tumor cells. It has been known that its extent is often enhanced in gastric cancer by some anti-cancer drugs, such as chemotherapeutic drugs, hormones or immune agents, micro-vascular growth inhibitors have been proved to have some inhibitory effects on maligant tumors,especially on gastric tumors. But it has not been clear whether NM-3 is a micro-vascular inhibitive agent for solid tumor growth[1-3], it might suppress gastric cancer cell proliferation and cause tumor cell loss and nuclear condensation in vitro. Up to date, NM-3 is considered as the newest micro-vasular inhabitor[1-4]. Combined with carboplatin, it can soften hard lumps and dissolve phlegm, enhance apoptosis of human gastric cancer xenografts in nude mice. On the other hand, NM-3 can enhance the sensitivity of chemotherapeutic drugs on human gastric cancer. Based on previous studies, NM-3 exerts its effects on solid tumor growth by promoting apoptosis of human gastric cancer cell SGC-7901 and increasing the suppressive effects of carboplatin.

MATERIALS AND METHODS

Materials

A human gastric cancer cell line SGC-7901 grafted onto nude mice was used as the animal model, the age of these 60 mice was 6-7 weeks old female balb/c-nu/nu mice (weight 18-22 g) and a human gastric cancer cell line SGC-7901 was obtained from Shanghai Tumor Institute (No: 01842). The animals were subcutaneously xerografted under abdominal skin with the SGC-7901 cell line. The tumor transplantation procedure was described previously. The animal model and SGC-7901 cell line were obtained from Shanghai Experimental Animal Centre, Chinese Academy of Sciences.

NM-3 was composed of 2-c8-hydroxy-6-methoxy-1-oxo-1 h-2-benzopyran-3-yd, concentrantion of NM-3 was 100 mg·L^-1, the concentration of carboplatin was 100 mg·L^-1.

Methods

Experimental schedule: After grafting, these nude mice were randomly divided into 3 groups: control group and two experimental groups assigned to receive NM-3 or carboplatin respectly. Each experimental mouse in two experimental groups was given a 0.5 mL dose of NM-3 drug via intra-abdominal injection or gastric perfusion (empty control group) once every three days over a 40-day period beginning at 1^st day after being xerografted. The control animals received normal saline according to the same schedule by gastric perfusion. The animals were killed 41 d after being xerografted.

In our study, NM-3 induced gastric cancer cell apoptosis, and enhanced the chemotherapeutic sensitivity of human gastric cancer cell line SGC-7901 on carboplatin in vitro. Apoptosis induced by NM-3 needed further investigation.

Therapeutic effects on human gastric cancer cell growth were assessed. Tumor size was measured twice a week by multiplying two perpendicular diameter and tumor weight was determined immediately by electron balance after the animals were killed. Apoptotic cells and apoptotic index were determined by the terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate-fluorescene nick end labeling (TUNEL) method and flow cytometry analysis. Morphological atterations were observed with electron microscope.

Flow cytometry analysis: Propidium iodide (PI) staining was used for flow cytometric detection of apoptosis. 1 × 10⁶ cells from each of the samples were treated with RNase and stained with PI. The apoptotic cells labled by DNA strand were measured with a flow cytometer (FACS Calibur,Becton Dickinson,U.S.A).The data from 1 × 10⁶ cells/sample were collected,stored,and analyzed using CELLQUES’T and MODFITLT for macV1.01 software[4-12].

Statistical analysis

The results were expressed as x^- ± s, Student’s t test was used. P value < 0.05 was concidered significantly.

RESULTS

NM-3 inhibited growth of micro-vascular of tumor in nude mice with human gastric cancer SGC-7901

NM-3 group decreased signicantly the neo-microvascular density (1.17 ± 0.05 mm³) of gastric cancer tumor implanted onto nude mice was significantly decreased in NM-3 group compared with that in saline group (5.37 ± 1.12 mm³) and carboplatin group (4.72 ± 1.18 mm³, P > 0.05). The micro-vascular density in NM-3 combinated with carboplatin group (1.18 ± 0.05 mm³) was not significantly different from that in NM-3 group (P > 0.05, Table 1).

Table 1.

Growth of neo-microvascular around gastric tumor supressed by NM-3 (x^- ± s)

Treatment	n	Density (mm3)
Carboplatin	10	4.72 ± 1.18a
NM-3 plus carboplatin	10	1.18 ± 0.05b
Saline	10	5.37 ± 1.72

^aP > 0.05,

^bP < 0.01 vs t test in saline control group.

NM-3 enhanced sensitivity of carboplatin on human gastric cancer induced by apoptosis of human gastric cancer cell in vitro or in vivo

The apoptotic indix (AI) of SGC-7901 induced by carboplatin was enhanced in NM-3 group. The apoptotic indix (TUNEL: 27.98% ± 6.12%, FACScan: 26.86% ± 5.69%) was markedly increasd in that of carboplatin group by using either TUNEL method or flow cytometry analysis compared with the carboplatin group (TUNEL: 12.94% ± 2.12%, P < 0.01; FACScan: 11.86% ± 1.09%, P < 0.01). The apoptotic indix in NM-3 group (TUNEL: 16.47% ± 4.13% FACScan: 15.97% ± 1.49%) was higher than that in normal saline group (TUNEL 1.83% ± 0.12%, P < 0.01; FACScan: 1.06% ± 0.09%, P < 0.01, Table 2).

Table 2.

Apoptotic indix (AI) of human gastric cancer line SGC-7901 enhanced by NM-3 in vitro (x^- ± s)

Treatment	n	AI (TUNEL)%	AI (FACScan)%
NM-3	10	16.47 ± 4.13b	15.97 ± 2.49b
Carboplatin	10	12.94 ± 2.12b	11.86 ± 1.09b
NM-3 plus carboplatin	10	27.98 ± 6.12b	26.86 ± 5.69b
Saline	10	1.83 ± 0.12	1.06 ± 0.09

^aP > 0.05,

^bP < 0.01 vs t test, in saline control group.

NM-3 effected on growth of xerografted human gastric cancer cell line SGC-7901 in nude mice

The tumor weight of nude mice in NM-3 group (4.17 ± 0.22 g) was obviously lower than that in normal saline controls (9.45 ± 0.38 g), the tumor size of the prior group (0.68 g ± 0.07cm³, P < 0.05) was smaller than that in normal saline control group (8.94 ± 1.46 cm³). The tumor weight and size of nude mice in NM-3 combinated with carboplatin group were 2.78 ± 0.18 g and 0.34 ± 0.02 cm³ respectively. However, those in NM-3 group were not significantly different compared with carboplatin group (4.46 ± 0.23 g, 0.71 ± 0.08 cm³) 7 weeks later. Tumor growth was (size and weight) markedly inhibited by treatment with NM-3 (P < 0.01). The tumor inhibitory rate of single NM-3 on tumors was 67.7%, that in NM-3 combinated with carboplatin group was up to 98.7% (Table 3).

Table 3.

Growth of xerografted human gastric tumor affected by NM-3 in nude mice (x^- ± s)

Treatment	n	Weight (g)	Size (cm3)
NM-3	10	4.17 ± 0.22a	0.68 ± 0.07a
Carboplatin	10	4.46 ± 0.23a	0.71 ± 0.08a
NM-3 plus carboplatin	10	2.78 ± 0.18b	0.34 ± 0.02b
Saline	10	9.45 ± 1.38	8.94 ± 1.46

^aP < 0.05,

^bP < 0.01 vs t test, in saline control group.

DISCUSSION

Gastric cancer remains one of the most common causes of cancer-related death in the world. At present, gastric cancer is still diagnosed at its advanced stage in most patients throughout the world. Even with curative resection, they remain at a high risk of relapse[13-32]. Thus, there is a great need for effective adjuvant therapy for patients with gastric cancer. Our previous clinic paired comparative studies suggested that NM-3 had therapeutic effects on advanced gastric cancer. It could increase the surviving period of the patients, improve the life quality and increase the metastasis and recurrence after operation because of its lower toxic side-effect compared with intravenous chemical therapy[33-47]. Up to date, the effect of NM-3 on human gastric cancer has not been reported in the world. So we thought it is worth to make a further research on its anti-cancer mechanisms.

Gastric cancer is not only a disease with abnormal cell proliferation and differentiation, but also a disease with abnormal apoptosis. Enhanced apoptosis in human gastric cancer cells could be observed after treatmemt with 5-fluorouracil, cisplatin, arsenous oxide, etc. These data suggest that it is a therapeutic method for patients with gastric cancer to induce apoptosis of cancer cells[64]. The present study indicated that tumor growth was significantly inhibited by treatment with carboplatin or NM-3. The results obtained by TUNEL method and cytometry analysis suggested that gastric cancer cells were suppressed in vivo, NM-3 was related to the induction of apoptosis of human gastric cancer cell line SGC-7901. These data suggest that NM-3 can inhibit gastric cell proliferation. So inhibition of gastric cancer induced by NM-3 is also related to the suppression of its proliferation.

Apoptosis is a complex and active cellular process, whereby individual cells are triggered to undergo self-destruction in a manner that would neither injures neghiboring cells nor elicites any inflammatory reation. Various triggering factors initiate corresponding proteo-lysis cascade reaction depending on mitochondrion or APO 1\FAS\CD95 receptors mediate apoptotic pathways. There are oncogenes and tumor suppressor gene products in the regulation and execution of apoptosis. It has been proved[14-22] that p53, Rb, myc, ras, raf, play important roles in apoptosis and are thus named the guardians of genomes[23-44]. They monitor the state of DNA and cell cycle is blocked in case of DNA damage. This takes place through the induction of CIP/Swaf/p21. In the absence of phosphorylated active cyclin-dependent kinases, the cell cycle remains inactive (unphosphorylated).

This leads to activation of DNA repair machinery. If DNA repair fails, p53 will take over again and trigger apoptosis in a process that involves upregulation of the apoptosis-inducing bax and down-regulation of the apoptotic bal-2[45-60]. We also detected apoptosis-inhibiting member of the bcl-2 family[61-64]: bcl-2 mRNA. The interaction between NM-3 and gastric cancer cell SGC-7901 induced its apoptosis of gastric cancer cells, but further in vivo or in vitro studies are needed.