| Gastric cancer remains one of the most common forms of cancer worldwide Around 9 million cases of gastric cancer are diagnosed worldwide each year, with the highest incidence occurring in eastern Asia, Eastern Europe, and the Andean regions of South America Furthermore, most gastric cancer patients have tumor recurrence after treatment In order to prevent recurrence after treatment, it is important to develop medicines which are high efficacy and new mechanisms of action. Noscapine is a phthalide isoquinoline non-narcotic alkaloid derived from opium,for more than 75 years, noscapine has traditionally been used as an oral cough suppressant with no know toxic side effects in human. Noscapine's chemical structure is similar to the known microtubule binding agent, colchicines, and its binding activity to microtubules has been extensively characterized. Noscapine hydrochloride is in phaseâ… /â…¡clinical trials for the treatment of chronic lymphocytic leukemia or low grade non Hodgkin's lymphoma refractory to chemotherapy and hematological malignancies. Noscapine binds stoichiometrically to tubulin and promotes microtubule polymerization, which causes growth arrest of tumor cells during mitosis. Moreover, noscapine anti-cancer activity involve induction of apoptosis via mitochondrial pathways was demonstrated in various cancers. So far, there is no available information about the antitumor effects of noscapine on human gastric cancer cells. In this study, we addressed the hypothesis that noscapine play an important role in mitochondria-mediated apoptosis in gastric cancer cells. To test our hypothesis, we investigated the mechanism of action, by which noscapine induces apoptosis in gastric cancer cell lines. We demonstrated that mitochondrial pathways involved noscapine-mediated apoptosis in gastric cancer cell lines. In vitro study, noscapine was used in four gastric cancer cell lines, BGC823,SGC7901,MGC803 and HGC27, changes and apoptosis of tumor cells was observed, its mechanism was preliminary explored. Secondly, in vivo study, gastric cancer cells xenograft model in nude mice subcutaneously was established, tumor inhibition of noscapine was observed. Finally, we observed tumor inhibition of noscapine combined lock-down CDH17 (Liver-intestine cadherin) gene. Based on the above findings, a potential new way of clinical treatment of gastric cancer was found.Methods1. Gastric cancer cells were treated with noscapine, survival and growth inhibition rate was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay; conditions of apoptosis were measured by Hoechst33258 and Annexin V-FITC/PI of Flow Cytomtry; activity of procaspase-3, procaspase-9, cleaved caspase-3 and cleaved caspase-9 were measured by Western blotting.2. Gastric cancer BGC823 cell,5×106, suspended in 100μL PBS, were subcutaneously inoculated into the lower right flank of the nude mice. When the tumors were 100-150 mmJ in size, noscapine (10,20 and 40mg/kg) were administrated via intratumoral injection every 3 days. Tumor growth was monitored using calipers every 3 days. The nude mice were killed at 33 days.Tumor volume (V) was calculated by using the formula:tumor volume V (mm3)=-Ï€/6×length (mm)×width (mm)2. Status of nude mice and inhibitory effect of tumor growth was observed, the tumor volume was calculated, and growth curve was plotted. Representative histological sections of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)were taken from mice bearing transplantation tumor were detected.3. Construction of CDH17 miRNA Lentiviruses, A polâ…¡expression system plasmid(Invitrogen, Carlsbad, CA, USA), which is based on the miRNA vector system and includes endogenous murine miR-155 flanking sequences and a spectinomycin resistance gene, was used for the construction of human CDH17 miRNA expression plasmids. Four precursor miRNA sequences (SRI-4) targeting to CDH17 (GenBank accession number NM004063)were designed using an Internet application system (Invitrogen, Carlsbad, USA). And a randomized sequence was construct as a negative-control. These double-stranded oligonucleotides were inserted into pcDNATM6.2-GW/EmGFP-miR expression vector (Invitrogen, Carlsbad, USA) and then transient transfected into BGC823 cells according to the operating manual of Lipofectamine 2000 (Invitrogen, Carlsbad, USA), then DNA sequencing were performed for confirming successful construction. RT-PCR and western blot showed that the SR4 yielded the best suppression efficiency (data not shown), therefore, The pDONRTM221 vector was used as an intermediate to transfer the pre-miRNA expression cassette into the lentiviral expression plasmid (pLenti6/V5-DEST) using Gateway Technology (Invitrogen, Carlsbad, USA). The new miRNA expression vectors (plenti-CDH17-miR) and packaging vectors were cotransfected into 293 FT cells with Lipofectamine2000 according to the manufacturer's instructions (Invitrogen) the viral supernatant was harvested 48 hours after transfection.BGC823 cells were transduced with specific (Lenti-CDH17-miR-B) or negative control lentiviral vectors (Lenti-CDH17-miR-neg, Mock) and selected for stable integrants by culturing a complete medium containing blasticidin for 2 weeks.Results1. After treatment of the four gastric cancer cell lines (BGC823, SGC7901, MGC803, HGC27) with 10μM noscapine, cell viability was determined by MTT asssy. noscapine significantly inhibited the proliferation of all cell lines tested. Especially, noscapine at 12 h,24 h and 36 h inhibited BGC823 cells viability by (10.26±2.10)% (21.71±3.51)%and (76.34±6.25)%of control group respectively. Furthermore, the number of BGC823 cells was greatly decreased by incubation 100μM and 150μM noscapine, while a minimal decrease was shown in 10μM noscapine after treatment for 24h.These data indicated that noscapine has a potential not only to prevent gastric cancer cell growth, but also to reduce the number of gastric cancer cells. In addition, the morphological changes of the apoptotic BGC823 cells were detected by Hoechst 33258 staining. Chromatin condensation and nuclear fragmentation are the classic characteristics of apoptotic cells. In the control cells, the nuclei were stained a weak homogeneous blue, while in treatment groups, bright chromatin condensation and nuclear fragmentation could be found. Apoptosis is characterized by distinct morphological features such as nuclear fragmentation and chromatin condensation. Noscapine has been reported to have an anti-tumor effect by apoptosis induction in various solid tumors. Thus, we examined here if it resulted in gastric cancer cell death via apoptosis. BGC823 cells were treated with noscapine (50-150μM) for 24h followed by annexin V/PI staining to examine the proportion of apoptotic cells. It revealed that noscapine caused cell apoptosis in a dose-dependent manner. The apoptosis is initiated by a caspase cascade. We therefore addressed whether noscapine induced apoptosis by caspase signaling. BGC823 cells were treated with 50μM, 100μM and 150μM noscapine for 24h, followed by Western blot to assess the caspase activation. Cleaved forms of caspase-3 and caspase-9 were detected, indicating that noscapine induced apoptosis via caspase cascade. Bax protein was upregulation and Bcl-2 protein was downregulation after noscapine treatment. It is established that Bcl-2 inhibits Bax activity, which reduces mitochondrial membrane potential, leading to cell apoptosis.2. Following the investigation of apoptosis induction in BGC823 cell in vitro, the anti-tumor effect of noscapine was evaluated. The in vivo study was investigated in xenograft model. After growing for 6 days, the tumor xenografts reached a mean size of 100 mm3. We chose 18 mice with tumor xenografts of around 100 mm3 in size and randomly divided them into three groups. There were no statistical differences among the sizes of all the groups. The nude mice were given different treatments. Noscapine cause the inhibitory effects on the growth of tumor in vivo. Control group, in which tumors grew progressively and reached 1000 mm3 within 18 days. However, the treatment groups were significantly suppressed. Treatment with noscapine reduced tumor growth in a dose-dependent manner. At the end of 33 days, noscapine at 10 mg/kg,20 mg/kg and 40mg/kg inhibited tumor growth by 45%,60%and 81%of control group (2178 mm) respectively (Fig.5 A). Compare with control group, tumor weight was only 0.206g in high dose group at the end of the experiment. TUNEL assay of the subcutaneous tumor sections demonstrated noscapine caused obvious cell death in tumor mass via apoptosis, whereas less apoptosis was found in control group (p< 0.05). These results proved that noscapine has significant anti-tumoral potential in vivo.3. The lentiviral vector expression cassette allowed for the permanent expression of GFP and CDH17 miRNA in transduced cells. To test the knockdown efficiency, we examined the expression of CDH17 on both mRNA and protein levels. Data showed that after being transfected with a MOI of 10 for 72 hours, the mRNA and protein levels of CDH17 were downregulated in lenti-CDH17-miR-B cells compared with BGC823 and lenti-CDH17-miR-neg cells. Downexpression of CHD17 gene can enhance apoptosis-inducing effects of noscapine on human gastric cancer BGC823 cells. Apoptosis was detected using flow cytometry and Hoechst33258 staining. The treatment with noscapine upregulated Bax protein, downregulated Bcl-2 and Bcl-xL protein. In addition, Cyt-c protein was upregulated, suggesting that the apoptosis is Bcl-dependent pathway. Moreover, in xenograft tumor mouse model, downexpression of CHD17 increased the efficacy of cisplatin-induced inhibition of tumor growth in nude mice via apoptosis induction which was demonstrated by TUNEL assay.ConclusionsThese data of the study suggest that noscapine induces apoptosis in gastric cancer cells via mitochondrial pathways in vitro and in vivo. Downexpression of extrinsic CHD17 gene can conspicuously ameliorate apoptosis-inducing effects of cisplatin on human gastric cancer BGC823 cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer. Based on the above findings, a potential new way of clinical treatment of colon cancer was found. |
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