Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

To investigate the effects of anti-sense peptide nucleic acids （PNAs） targeting Ki-67 gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations （1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L）. The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and ^3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide （ASODNs） targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs （16.9±0.7） was significantly inhibited as compared with that of the control groups （28.6±0.4） （P〈0.01）. The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs （42.1 ±2.2） was significantly reduced when compared with that of the control groups （83.6± 1.4） （P〈0.01）. Proliferation of 786-0 cells treated with anti-sense PNAs （20.7 ± 1.5） was significantly inhibited as compared with that of the control groups （58.6± 1.4） （P〈0.01）. The apoptosis rate of 786-0 cells treated with anti-sense PNAs （28.7 ± 2.3） was significantly increased higher compared with that of the control groups （13.8 ±1.0） （P〈0.01）. From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Overexpression of p27^(KIP1)induced cell cycle arrest in G_1 phase and subsequent apoptosis in HCC-9204 cell line

AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.

Growth inhibition and apoptosis induction Sulindac on Human gastric cancer cells

AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC) cells. METHODS: The human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG(2) and SMMC7721 were used for the study. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygenase-2 (COX-2) and Bcl-2 were detected by Western dot blotting. RESULTS: Sulindac could initiate growth inhibition and apoptosis of MKN45, MKN28, HepG(2) and SMMC7721 cells in a dose-and time-dependent manner. Growth inhibitory activity and apoptosis were more sensitive in HepG(2) cells than in SMMC7721 cells, MKN45 and MKN28 cells. After 24 hours incubation with sulindac at 2mmol x L(-1) and 4mmol x L(-1), the level of COX-2 and Bcl-2 protein were lowered in MKN45, SMMC7721 and HepG(2) cells but not in MKN28 cells. CONCLUSION: Sulindac could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2 and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater than that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells.

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells

AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.

Neuroendocrine neoplasms of liver-A 5-year retrospective clinico-pathological study applying World Health Organization 2010 classification

AIM To study the clinicopathological characteristics of neuroendocrine neoplasms(NEN) on liver samples and apply World Health Organization(WHO) 2010 grading of gastroenteropancreatic(GEP) NEN.METHODS Clinicopathological features of 79 cases of NEN of the liver diagnosed between January 2011 to December 2015 were analyzed. WHO 2010 classification of GEP NEN was applied and the tumors were graded as G1, G2 or G3. Two more categories, D1/2(discordant 1/2) and D2/3(discordant 2/3) were also applied. The D1/2 grade tumors had a mitotic count of G1 and Ki-67 index of G2. The D2/3 tumors had a mitotic count of G2 and Ki-67 index of G3. The follow up details which were available till the end of the study period(December 2015) were collected.RESULTS Of the 79 tumors, 16 each were G1 and G2, and 18 were G3 tumors. Of the remaining 29 tumors, 13 were assigned to D1/2 and 16 were D2/3 grade. Male preponderance was noted in all tumors except for G2 neoplasms, which showed a slight female predilection. The median age at presentation was 47 years(range 10-82 years). The most common presentation was abdominal pain(81%). Pancreas(49%) was the most common site of primary followed by gastrointestinal tract(24.4%) and lungs(18%). Radiologically, 87% of the patients had multiple liver lesions. Histopathologically, necrosis was seen in only D2/3 and G3 tumors. Microvascular invasion was seen in all grades. Metastasis occurred in all grades of primary NEN and the grades of the metastatic tumors and their corresponding primary tumors were similar in 67% of the cases. Of the 79 patients, 36 had at least one follow up visit with a median duration of follow up of 8.5 mo(range: 1-50 mo). This study did not show any impact of the grade of tumor on the short term clinical outcome of these patients.CONCLUSION Liver biopsy is an important tool for clinicopathological characterization and grading of NEN, especially when the primary is not identified. Eighty-seven percent of the patients had multifocal liver lesions irrespective of the WHO grade, indicating a higher stage of disease at presentation. Follow up duration was inadequate to derive any meaningful conclusion on long term outcome in our study patients.

Influence of norcantharidin on proliferation,proliferation-related gene proteins prolifera-ting cell nuclear antigen and Ki-67 of human gallbladder carcinoma GBC-SD cells

BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.

进展期胃癌术中组织间注射靶向化疗对转移淋巴结Ki-67表达及细胞凋亡的影响

目的:了解进展期胃癌术中组织间注射靶向化疗(IICT)对转移淋巴结Ki-67表达和细胞凋亡的影响。方法:将满足要求的28例进展期胃癌病例随机分成治疗组和对照组。治疗组先于肿瘤组织及周围注射亚甲蓝和丝裂霉素的混合液后再手术,对照组直接手术。观察两组病例转移淋巴结的肿瘤坏死变性有效性、Ki-67表达和细胞凋亡情况。结果:治疗组转移淋巴结的肿瘤细胞变性坏死有效率高于对照组,差异有统计学意义(P<0.05);治疗组治疗后转移淋巴结Ki-67表达阳性率低于治疗前,肿瘤细胞凋亡指数高于治疗前,差异均有统计学意义(P<0.05);治疗组治疗后转移淋巴结的Ki-67表达低于对照组,肿瘤细胞凋亡指数高于对照组,差异有统计学意义(P<0.05).结论:IICT能够抑制胃癌转移淋巴结内肿瘤细胞增殖,促进肿瘤细胞凋亡,对防止医源性扩散、微转移、微残留和术后复发、转移可能有一定作用。

新辅助化疗后乳腺癌组织中survivin表达及其与Ki-67、AI的相关性研究

目的探讨采用CTF方案新辅助化疗后乳腺癌组织中凋亡抑制蛋白survivin的表达及其与肿瘤细胞凋亡、增殖的相互关系和临床意义。方法采用免疫组化SABC法,检测60例行新辅助化疗和60例未行新辅助化疗的乳腺癌组织中survivin和Ki-67的表达,并采用原位细胞末端转移标记法(TUNEL法)测定细胞凋亡指数(AI)。结果新辅助化疗组survivin阳性表达率为36.7%(22/60),明显低于对照组(71.7%,43/60)(χ2=14.821,P<0.001)。新辅助化疗组Ki-67阳性表达率(38.3%,23/60)明显低于对照组(61.7%,37/60,χ2=6.533,P<0.05)。新辅助化疗组AI均数为(9.34±3.12)%,对照组AI均数为(5.27±3.16)%,两组比较差异有统计学意义(γ=1.998,P<0.05)。新辅助化疗组survivin表达与AI呈负相关(γ=-0.36,P<0.05),而与Ki-67表达呈正相关(γ=+0.47,P<0.05)。新辅助化疗组化疗总有效率为73.3%(44/60),化疗后部分缓解(Ⅱ级)病例survivin表达水平(18.2%,8/44)明显低于无效(Ⅲ级)病例(81.3%,13/16)(χ2=20.514,P<0.001)。结论应用CTF方案新辅助化疗,缓解率高,近期疗效明显。Survivin表达水平可作为预测乳腺癌新辅助化疗疗效的指标。

CyclinD1和Ki-67在茉莉酸甲酯抑制人肝癌细胞HepG2增殖作用中的表达变化

目的:探讨CyclinD1、Ki-67蛋白在茉莉酸甲酯(MeJA)抑制人肝癌细胞HepG2细胞增殖过程中的表达变化。方法:免疫细胞化学检测HepG2细胞CyclinD1、Ki-67蛋白的表达。结果:MeJA作用HepG2细胞48 h后,CyclinD1、Ki-67蛋白表达水平明显下降(P<0.01)。结论:MeJA通过下调Cy-clinD1、Ki-67蛋白表达,使细胞周期相关蛋白的表达发生改变,减少处于增殖期的细胞数目,从而发挥抗肝癌作用。

The predictive value of vascular endothelial growth factor and Ki-67 expression on neoadjuvant therapy in rectal cancer

Objective： To investigate the expressions of vascular endothelial growth factor （VEGF） and proliferating cell nuclear antigen （Ki-67） in patients with rectal adenocarcinoma and their associations with neoadjuvant therapy. Methods： The expressions of Ki-67 and VEGF in 32 cases of rectal adenocarcinoma, including both pretreatment tumor biopsies and postoperative specimen, were detected by immunohistochemistry using specific antibodies, and were correlated with clinicopathological factors. Results： The intensity of VEGF staining was significantly correlated with lymph nodal metastasis （P =0.033）, depth of tumor invasion （P =0.007） and tumor stage （P= 0.016）, but not with histological types, tumor sizes, patients＇ ages and genders （P 〉 0.05）. Low level of VEGF expression had significant correlation with the high sensitivity of response to neoadjuvant therapy （P = 0.016）. The transient increase of VEGF expression could be seen after neoadjuvant therapy （P = 0.035）. Ki-67 labeling index （Ki-67-LI） was significantly correlated with lymph node metastasis （P = 0.028）, but not correlated to tumor sizes, patients＇ ages and genders （P 〉 0.05）. Tumors with lower Ki-67-LI were more sensitive to neoadjuvant therapy （P = 0.032）. The Ki-67 level decreased after neoadjuvant therapy, but no statistical significance was found （P 〉 0.05）. Conclusion： Our results demonstrate that the expression of VEGF and Ki-67 in pretreatment rectal adenocarcinoma biopsies may be predictive of tumor response to neoadjuvant therapy.

As_2O_3联合γ分泌酶抑制剂MW167对人肝癌HepG_2/ADM细胞耐药性的影响

目的:探讨三氧化二砷(As_2O_3)和γ分泌酶抑制剂MW167单独及联合作用对HepG_2/ADM细胞耐药性的影响,并阐明其作用机制。方法:MTT法检测不同浓度As_2O_3(0.25、0.50、1.00、2.00、4.00和8.00mg·L-1)和MW167(10、20和40μmol·L-1)处理HepG_2/ADM细胞24、48和72h后细胞的吸光度(A)值,计算细胞增殖抑制率,确定药物作用浓度和时间;将HepG_2/ADM细胞分为空白组、As_2O_3组、MW167组、As_2O_3和MW167联合组;根据MTT结果选取无细胞毒剂量的As_2O_3、MW167干预各组细胞48h后,FCM法检测细胞凋亡率;RT-PCR和Western blotting法分别检测各组细胞中Notch-1、Hes-1、MDR-1(P-gp)、Bcl-2和Bax mRNA及蛋白表达水平。结果:在同一浓度时,As_2O_3和MW167对HepG_2/ADM细胞的增殖抑制率随着作用时间的延长而升高(P<0.05或P<0.01);在同一时间点,As_2O_3和MW167对HepG_2/ADM细胞的增殖抑制率随药物浓度的增加而升高(P<0.05);确定As_2O_3和MW167的无毒剂量分别为0.25mg·L-1和10μmol·L-1,干预时间为48h。药物干预48h后,与空白组比较,各干预组细胞凋亡率均升高(P<0.05),其中以联合组升高最为明显(P<0.01)。与空白组比较,各干预组细胞中Notch-1、Hes-1、MDR-1(P-gp)和Bcl-2mRNA及蛋白表达水平明显降低(P<0.05),其中以联合组降低最为明显(P<0.01);与空白组比较,各干预组Bax mRNA和蛋白表达水平明显升高(P<0.05),其中以联合组升高最为明显(P<0.01)。结论:As_2O_3可逆转HepG_2/ADM细胞的耐药性,其作用机制可能与抑制细胞增殖、促进细胞凋亡、下调细胞中Notch-1、Hes-1、MDR-1(P-gp)和Bcl-2基因及蛋白表达水平及上调Bax基因和蛋白表达水平有关。

The prognostic molecular markers in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

Effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells

AIM: To investigate the effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells in vitro and its anticancer mechanism. METHODS: Human gallbladder carcinoma GBC-SD cells were cultured by cell culture technique. The growth and the invasiveness of GBC-SD cells in vitro were evaluated by the tetrazolium-based colorimetric assay and by the Matrigel experiment and the crossing-river test. Expression of PCNA, Ki-67, MMP2 and TIMP2 proteins of GBC-SD cells was determined by streptavidin-biotin complex method. RESULTS: In vitro norcantharidin inhibited the growth and proliferation of GBC-SD cells in a dose- and time-dependent manner, with the IC50 value of 56.18 μ/mL at 48 h. Norcantharidin began to inhibit the invasion of GBC-SD cells at the concentration of 5 μg/mL, and the invasive action of GBC-SD cells was inhibited completely and their crossing-river time was prolonged significantly at 40 μg/mL. After treatment with norcantharidin, the expression of PCNA, Ki-67, and MMP2 was significantly decreased. With the increase in TIMP2 expression, the MMP2 to TIMP2 ratio was decreased significantly (P<0.05). CONCLUSION: Norcantharidin inhibits the proliferation and growth of human gallbladder carcinoma cells in vitro at relatively low concentrations by inhibiting PCNA and Ki-67 expression. Its anti-invasive activity may be the result of decrease in MMP2 to TIMP2 ratio and reduced cell motility.

Inhibitory effect of tumor suppressor p33^(ING1b) and its synergy with p53 gene in hepatocellular carcinoma

AIM: To investigate the inhibitory effect of tumor suppressor p33ING1b and its synergy with p53 gene in hepatocellular carcinoma (HCC).METHODS: Recombinant sense and antisense p33ING1b plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lineswith different endogenous p53 gene expressions, the synergistic effect of p33ING1b with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21WAF1/CIP1. In addition, the expression and mutation rates of p33ING1b in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP).RESULTS: Overexpression of p33ING1b inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33ING1b and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21WAF1/CIP1. Immunostaining results showed co-localized P33ING1b with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P＜0.01).Among 28 HCC samples, p33ING1b presented a low gene mutation rate (7.1%).CONCLUSION: p33ING1b collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33ING1b normal function may be an important mechanism for the development of HCC retaining wildtype p53.

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.

扶正软坚抗癌方调控Akt/MDM2/P53信号通路对肝癌HepG2细胞恶性生物学行为的影响

目的:探讨扶正软坚抗癌方调控蛋白激酶B(Akt)/鼠双微体2(MDM2)/P53信号通路对肝癌HepG2细胞恶性生物学行为的影响。方法:采用0、0.05、0.10、0.20、0.40、0.80、1.60、3.20和6.40 g·mL^(-1)扶正软坚抗癌方分别处理HepG2细胞48 h,CCK-8法检测HepG2细胞存活率,筛选扶正软坚抗癌方浓度用于后续实验。将HepG2细胞分为对照组、低剂量扶正软坚抗癌方组(0.2 g·mL^(-1))、中剂量扶正软坚抗癌方组(0.4 g·mL^(-1))、高剂量扶正软坚抗癌方组(0.8 g·mL^(-1))、SC79组(8 mg·L^(-1)SC79)和高剂量扶正软坚抗癌方+SC79组(0.8 g·mL^(-1)扶正软坚抗癌方+8 mg·L^(-1)SC79)。CCK-8法检测各组HepG2细胞增殖活性,克隆形成实验检测各组HepG2细胞克隆形成率,流式细胞术检测各组HepG2细胞凋亡率,Transwell小室实验检测各组HepG2细胞迁移和侵袭细胞数,Western blotting法检测各组HepG2细胞中增殖细胞核抗原(PCNA)、含半胱氨酸的天冬氨酸蛋白酶3(Caspase-3)、基质金属蛋白酶(MMP)-2、MMP-9、磷酸化Akt(p-Akt)、磷酸化MDM2(p-MDM2)和P53蛋白表达水平。结果:随着扶正软坚抗癌方浓度(0、0.05、0.10、0.20、0.40、0.80、1.60、3.20和6.40 g·mL^(-1))的升高,HepG2细胞存活率逐渐降低(P<0.05),选取0.2、0.4和0.8 g·mL^(-1)扶正软坚抗癌方用于后续实验。CCK-8法检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞增殖活性均明显降低(P<0.05),并呈剂量依赖性,SC79组HepG2细胞增殖活性明显升高(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞增殖活性明显升高(P<0.05)。克隆形成实验检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞克隆形成率均明显降低(P<0.05),并呈剂量依赖性,SC79组HepG2细胞克隆形成率明显升高(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞克隆形成率明显升高(P<0.05)。流式细胞术检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞凋亡率均明显降低(P<0.05),并呈剂量依赖性,SC79组HepG2细胞凋亡率明显升高(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞凋亡率明显升高(P<0.05)。Transwell小室实验检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞迁移和侵袭细胞数均明显降低(P<0.05),并呈剂量依赖性,SC79组HepG2细胞迁移和侵袭细胞数均明显升高(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞迁移和侵袭细胞数均明显升高(P<0.05)。Western blotting法检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞中PCNA、MMP-2、MMP-9、p-Akt和p-MDM2蛋白表达水平均明显降低(P<0.05),并呈剂量依赖性;Caspase-3和P53蛋白表达水平均明显升高(P<0.05),并呈剂量依赖性;SC79组HepG2细胞中PCNA、MMP-2、MMP-9、p-Akt和p-MDM2蛋白表达水平均明显升高(P<0.05),Caspase-3和P53蛋白表达水平均明显降低(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞中PCNA、MMP-2、MMP-9、p-Akt和p-MDM2蛋白表达水平均明显升高(P<0.05),Caspase-3和P53蛋白表达水平均明显降低(P<0.05)。结论:扶正软坚抗癌方抑制HepG2细胞增殖、迁移和侵袭,促进细胞凋亡,其作用机制与抑制Akt/MDM2信号通路、上调P53蛋白表达有关。

载脂蛋白C1表达对人肝癌HepG2细胞增殖和凋亡的影响及其机制

目的:探讨载脂蛋白C1 (APOC1)表达对肝癌细胞增殖和凋亡的影响,并初步阐明其相关分子机制。方法:通过癌症基因组图谱(TCGA)数据库分析肝癌患者癌组织中APOC1 mRNA表达水平及其与患者预后的关系。采用实时荧光定量PCR (RT-qPCR)法检测不同肝癌细胞中APOC1mRNA表达水平,筛选APOC1低表达的人肝癌HepG2细胞作为研究对象。将pcDNA3.1-APOC1质粒转染至HepG2细胞过表达APOC1 (APOC1过表达组),以转染空载体pcDNA3.1的HepG2细胞为对照组,采用MTS法和5-乙炔基-2'-脱氧尿嘧啶核苷(EdU)染色法检测2组细胞增殖活性和增殖率,Transwell小室实验检测2组细胞中迁移细胞数,流式细胞术和TUNEL法检测2组不同细胞周期细胞百分率和细胞凋亡率,Western blotting法检测2组细胞中细胞外调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)、蛋白激酶B (AKT)、磷酸化AKT (p-AKT)、B细胞淋巴瘤2 (Bcl-2)和活化型含半胱氨酸的天冬氨酸蛋白水解酶3 (cleaved caspase-3)蛋白表达水平。结果:TCGA数据库分析,肝癌患者癌组织中APOC1 mRNA表达水平低于正常肝组织(P<0.05),并且APOC1 mRNA低表达组肝癌患者预后较差。RT-qPCR法检测,HepG2细胞中APOC1 mRNA表达水平最低,选取该细胞作为后续研究对象。与对照组比较,APOC1过表达组细胞增殖活性和增殖率明显降低(P<0.05或P<0.01),迁移细胞数明显减少(P<0.01), S期细胞百分率和细胞凋亡率明显升高(P<0.01)。与对照组比较,APOC1过表达组细胞中p-ERK、 p-AKT和Bcl-2蛋白表达水平明显降低(P<0.05),cleaved caspase-3蛋白表达水平明显升高(P<0.01)。结论:APOC1高表达能够抑制人肝癌HepG2细胞的增殖,并诱导细胞凋亡,其机制可能与其抑制p-ERK、p-AKT、Bcl-2蛋白表达和促进cleaved caspase-3蛋白表达有关。

健脾理气药诱导人肝癌细胞SMMC7721凋亡的研究

目的观察健脾理气药的诱导凋亡效应,为其临床应用进一步提供依据。方法采用血清药理学方法研究中药的体外效应,应用四甲基偶氮唑蓝(MTT)比色法检测含中药兔血清对肝癌细胞的抑制效应,以Annexin V标记法、DNA含量测定、电子显微镜方法检测含中药血清诱导凋亡及细胞周期阻滞效应。免疫组化法观察含中药血清对P53,P21^(WAF1)/CIP1)蛋白的影响,RT-PCR法观察含中药血清对P21^(WAF1/CIP1) mRNA表达水平的影响。结果含中药血清有一定的抑制肝癌细胞作用,其作用3d的抑制率为6.6％,作用6d的抑制率为36.2％。含中药血清作用2d诱导9.8％±4.0％的肝癌细胞凋亡,使细胞周期阻滞于S期,并上调P53蛋白、P21^(WAF1/CIP1) mRNA及蛋白的表达。结论健脾理气药具一定的诱导凋亡及抑制肝癌细胞效应,上调p53,p21^(WAF1/CIP1)基因的表达为分子机制。

肝细胞癌中microRNA对细胞凋亡、转移和周期的调控作用

microRNA(miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子,参与转录后基因表达调控。大量证据显示一些miRNA在肝细胞癌(HCC)中表达失调,且发现miRNA通过一些通路或基因调控癌细胞的增殖、凋亡、转移、侵袭和细胞周期。另外,也有一些研究认为miRNA是癌基因或抑癌基因。因此,深入的了解miRNA在HCC中的具体作用和功能,或许会为HCC的治疗提供新的途径。