A novel cromakalim analogue induces cell cycle arrest and apoptosis in human cervical carcinoma HeLa cells through the caspase- and mitochondria-dependent pathway

In the present study, a series of seven synthetic croma-kalim analogues were prepared and evaluated for cytotoxic effect on human cervical carcinoma HeLa cells using WST-8 assay. A preliminary screening of these cromakalim analogues showed that 1-[(3S,4R)-4-(2-ethoxy-4-methyl-1H-pyrrol-1-yl)-3-hydroxy- 2,2-dimethylchroman-6-yl-3-phenylurea (compound?6) had the highest cytotoxic effect (IC50 of 138 μM) and significantly inhibited HeLa cell proliferation after 36 h. In an effort to understand the cytotoxic mechanism of compound?6, we examined its effect on apoptosis and cell cycle distribution. Our results showed that compound 6 induced marked changes in apoptotic morphology and significantly increased early apoptosis of HeLa cells after 48 h by using Annexin V-FITC/PI dual staining assay. This apoptotic induction was associated with an increase in Bax expression, a decrease in Bcl-2 expression, release of cytochrome c and subsequent activation of caspase-9 and -3, which indicated that compound 6 induced apoptosis via caspase- and mitochondria-dependent pathway. By DNA content analysis and [3H]thymidine incorporation assay, compound 6 was found to induce an increase in the number of cells in G1 phase, accompanied by a decrease in the S phase to prevent DNA synthesis after 24 h of treatment. In addition, compound 6 caused significant DNA damage, as detected by the alkaline comet assay. Taken together, the data demonstrate that compound 6 induces apoptosis in HeLa cells through caspase- and mitochondria-dependent pathway and this apoptotic effect is associated with cell cycle arrest and DNA damage. These findings provide further understanding of the molecular mechanisms of compound 6 in cervical cancer.     

Selenomethionine induces p53 mediated cell cycle arrest and apoptosis in human colon cancer cells

While there is an increasing interest in selenium chemoprevention against human colon polyp recurrence and other cancers, the mechanism(s) by which these agents inhibit carcinogenesis are uncertain. Some of the proposed mechanisms include the inhibition of cytosine methyltransferases, carcinogen bioactivation, and inhibition of cyclooxygenase (COX). More recently, it has been suggested that selenium may exert growth inhibitory effects by activating p53. However, the molecular mechanisms of action of selenomethionine, an organoselenium compound present in selenized yeast and currently being investigated in human clinical trials for colon polyp prevention, are unclear. In the present study we tested the hypothesis that selenomethionine might affect colon cancer cell growth by p53 mediated apoptosis and/or cell cycle regulation. Four human colon cancer cell lines including HCT116 and RKO (wild type p53), HCT116-p53KO (isogenic control of HCT116 cells with p53 knocked out) and Caco-2 (mutant p53) were treated with 0-100 microM of selenomethionine for 24, 48 and 72 h. Cell viability rates were determined by the MTT assay. Cell cycle analysis was performed by flow cytometry and apoptosis measured by Annexin V-Cy5 staining. Expression of p53 protein was determined by Western blotting and immunofluorescence assays. All cell lines showed concentration and time dependent growth inhibition with selenomethionine, although HCT116 and RKO cells were the most sensitive to such treatments. Interestingly, although HCT116 and HCT116-p53KO are isogenic cell lines, selenomethionine caused a G2/M cell cycle arrest in HCT116 and RKO cells, but not in HCT116-p53KO cells. Similarly, both HCT116 and RKO demonstrated a significant increase in apoptosis (100-170%; p < 0.01) with 50-100 microM selenomethionine. Cell cycle arrest and apoptosis observed in HCT116 and RKO cell lines were accompanied by a marked increase in p53 protein expression following selenium treatment. These results clearly suggest that selenomethionine exerts p53 dependent growth inhibitory effects in colon cancer cells by inducing G2/M cell cycle arrest as well as apoptosis.     

2'-Nitroflavone induces cell cycle arrest and apoptosis in HeLa human cervical carcinoma cells

The mechanism of antitumor action of a synthetic nitroflavone derivative, 2'-nitroflavone, was evaluated in vitro in HeLa human cervix adenocarcinoma cells. We showed that the nitroflavone derivative slowed down the cell cycle at the S phase and increase the population of cells at the G(2)/M phase after 24h of incubation. The treatment with 2'-nitroflavone also induced an apoptotic response, characterized by an increase of the sub-G1 fraction of cells, by cells with chromatin condensation and membrane blebbing, by a typical ladder of DNA fragmentation and by detection of apoptotic cells stained with Annexin V. The observed apoptosis was regulated by caspase-8 and -9, both contributing to the activation of the effector caspase-3. In addition, inhibitors of caspase-8 or -9 partially protected HeLa cells from 2'-nitroflavone-induced cell death. We also found that 2'-nitroflavone did not affect the total amount of Bax and Bcl-2 proteins, although a translocation of Bax from cytosol to mitochondria was evident after 6h of exposure. Furthermore, 2'-nitroflavone decreased the expression of the anti-apoptotic Bcl-X(L) protein, induced the release of cytochrome C to cytosol and increased the levels of Fas and Fas-L. Our results indicated that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2'-nitroflavone and suggest that this derivative could be a potentially useful agent for the treatment of certain malignancies.     

Phenethyl isothiocyanate (PEITC) promotes G2/M phase arrest via p53 expression and induces apoptosis through caspase- and mitochondria-dependent signaling pathways in human prostate cancer DU 145 cells

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.     

Cantharidin induces apoptosis in human bladder cancer TSGH 8301 cells through mitochondria-dependent signal pathways

Cantharidin has shown potent anticancer activities on many types of human cancer cells. This study was performed to elucidate whether mitochondria and caspases are involved in the modulation of apoptosis and cell cycle arrest by cantharidin in human bladder cancer cells. The effect of cantharidin on cell cycle arrest, apoptosis, caspases, reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ(m)) were measured by flow cytometry, and the levels of apoptosis-associated proteins and its regulatory molecules were studied by Western blotting. Cantharidin-induced apoptosis and DNA damage was determined by flow cytometric analysis, DAPI staining and Comet assay. After cantharidin treatment, the active forms of caspase-3, -8 and -9 were promoted. Cantharidin-induced apoptosis was associated with enhanced ROS and Ca(2+) generations, caused DNA damage, decreased the levels of ΔΨ(m) and promoted Endo G and AIF released from mitochondria. Cantharidin-induced G0/G1 arrest was associated with a marked decrease in the protein expressions of cyclin E and Cdc25c but promoted the levels of p21 and p-p53. Cantharidin-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and PARP, but down-regulation of the protein levels of Bcl-2, resulting in dysfunction of mitochondria then led to Endo G and AIF release for causing induction of apoptosis.     

Ursolic acid induces apoptosis through caspase-3 activation and cell cycle arrest in HaCat cells

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many kinds of medicinal plants and present in human diet. In this study, we investigated the pro-apoptotic effect of UA on HaCat derived keratinocyte cell line. Treatment with UA decreased the viability of HaCat cells in a concentration- and time-dependent manner. In addition, cell cycle analysis revealed that UA treated HaCat cells were blocked predominantly in G1 phase. Moreover, expression of p21WAF1, a cell cycle regulator, was increased by UA, indicating that UA-induced cell cycle arrest could be mediated through p21WAF1. During UA treatment, we also demonstrated that p53 was phosphorylated at serine 392 and translocated to the nucleus. It is well established that p53 achieves its tumor suppressor activity by inducing apoptosis on cells. To define the apoptotic process in our system, we examined effect of UA on caspase activities, and demonstrated caspase-3 activation. In conclusion, our results suggest that UA induces: i) cell cycle arrest concomitantly with the apparition of the apoptotic sub group G1 peak, and ii) cell death through apoptosis, which is mediated by caspase-3.     

TRICHOSTATIN A INHIBITS PROLIFERATION, INDUCES APOPTOSIS AND CELL CYCLE ARREST IN HELA CELLS

Objective： The histone deacetylase inhibitors （HDACIS） have been shown to inhibit cancer cell proliferation, stimulate apoptosis, an induce cell cycle arrest. Our purpose was to investigate the antiproliferative effects of a HDACI, trichostatin A （TSA）, against human cervical cancer cells （HeLa）. Methods： HeLa cells were treated in vitro with various concentrations of TSA. The inhibitory effect of TSA on the growth of HeLa cells was measured by MTT assay. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of TSA. Induction of cell cycle arrest was studied by flow cytometry. Changes in gene expression of p53, p21wafl and p27Kipl were studied by semiquantitative RT-PCR. Results： TSA inhibited cell growth in a time- and dose-dependent manner. Hoechst 33258 staining assay showed that TSA induced apoptosis. Cell cycle analysis indicated that treatment with TSA decreased the proportion of cells in S phase and increased the proportion of cells in G0/G1 and/or G2/M phases of the cell cycle. This was concomitant with overexpression of genes related to malignant phenotype, including an increase in p53, p21wall and p27Kipl. Conclusion： These results suggest that TSA is effective in inhibiting growth of HeLa cells in vitro. The findings raise the possibility that TSA may prove particularly effective in treatment of cervical cancers.     

二烯丙基三硫通过Caspase-3途径诱导人胃癌MGC803细胞凋亡

背景与目的:大蒜及其烯丙基硫化物具有抗肿瘤作用,但其具体的作用机制尚不十分清楚。本研究探讨二烯丙基三硫(diallyltrisulfide,DATS)诱导人胃癌MGC803细胞凋亡及凋亡过程中Caspase-3的变化及其意义。方法:采用荧光显微镜和电子显微镜观察以及流式细胞术和Westernblot检测等方法,观察DATS诱导MGC803细胞凋亡的作用及对活化的Caspase-3的影响。结果:在荧光显微镜及电子显微镜下,DATS作用的胃癌细胞出现了典型的凋亡形态学改变;流式细胞术检测出现明显的亚G1峰;12mg/L的DATS处理MGC803细胞0、4、8、12、24h后,活化的Caspase-3的表达率分别为1.9%、3.0%、7.3%、14.4%、27.6%,提示DATS呈时间依赖性促进活化的Caspase-3的表达;用特异的Caspase-3抑制剂预处理细胞后,再用12mg/LDATS处理细胞24h,与单用DATS组相比,凋亡率从23.0%降低到5.1%(P<0.01),提示Caspase-3的抑制剂能抑制DATS诱导的胃癌MGC803细胞凋亡。Westernblot显示酶原形式的Caspase-3被水解活化。结论:DATS诱导人胃癌MGC803细胞凋亡可能与活化Caspase-3有关。     

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and apoptosis in colorectal cancer cells via p53-dependent and -independent pathways

Many chemotherapeutic agents induce apoptosis via a p53-dependent pathway. However, up to 50% of human cancers have p53 mutation and loss of p53 function. Histone deacetylase inhibitors (HDACIs) are emerging as a potentially important new class of anticancer agents. Here, we report that, Trichostatin A (TSA), a pan-HDAC inhibitor, could induce G2/M cell cycle arrest and apoptosis in both colorectal cancer cell lines with wild-type p53 (HT116 cells) and mutant p53 (HT29 cells), although HCT116 cells had more apoptotic cells than HT29 cells. TSA induces apoptosis in both cell lines via the mitochondrial pathway as indicated by decrease of the mitochondrial membrane potential (MMP) and activation of caspase-3. Additionally, TSA induces expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in both cell lines. Bax knockdown by siRNA significantly impaired TSA-induced apoptosis in both cell lines. These data suggest that TSA induces G2/M cell cycle arrest and Bax-dependent apoptosis in colorectal cancer cells (HCT116 cells and HT29 cells) by both p53-dependent and -independent mechanisms. However, cells with normal p53 function are more sensitive to TSA-induced apoptosis.     

Reversine induces cell cycle arrest,polyploidy, and apoptosis in human breast cancer cells

Background

Reversine, a small synthetic purine analogue, has been reported to be effective in tumor suppression. In the present study, we demonstrated an antitumor activity of reversine that could suppress cellular proliferation and induce cell cycle arrest and apoptosis in human breast cancer cell lines.

Methods

To evaluate whether reversine could suppress cell growth of MCF-7 and MDA-MB-231 cells and induce cell death, the cell viability, cell cycle, and apoptosis were determined in this study.

Results

Reversine treatment in human breast cancer cells reduced cell viability in a dose-dependent manner. Cell cycle accumulation at the G₂/M phase in reversine-treated cells was also determined. Moreover, polyploidy was also found in reversine-treated cells. Apoptosis in reversine-treated cells was exhibited with PARP cleavage and caspase-3 and caspase-8 activation, but not caspase-9 activation, indicating that caspase-dependent apoptosis mediated by an extrinsic pathway took place in reversine-treated cells. Furthermore, reversine attenuated cell death in cells pretreated with a pan-caspase inhibitor before reversine treatment.

Conclusions

In the present study, we demonstrated that reversine contributes to growth inhibition in human breast cancer cells through cell cycle arrest, polyploidy, and/or apoptosis induction. The apoptosis mediated by reversine was induced by the mitochondria-independent pathway. Therefore, the potential role of reversine as a novel therapeutic agent for the treatment of breast cancer is worthy of further investigation.     

CR229, a novel derivative of beta-carbolin-1-one, induces cell cycle arrest and apoptosis in HeLa cells via p53 activation

In the course of screening for novel anticancer compounds, CR229 (6-Bromo-2,3,4,9-tetrahydro-carbolin-1-one), a novel derivative of beta-carbolin-1-one, was generated as a new scaffold candidate. For the first time, the authors demonstrate that CR229 inhibited the growth of HeLa cells by the induction of cell cycle arrest and apoptosis. Analysis of flow cytometry and western blots of HeLa cells treated with 2.5 microM CR229 revealed an appreciable cell cycle arrest in the G1, G2/M phase and apoptotic induction via the p53-dependent pathway. Furthermore, the release of cytochrome c from mitochondria was detected using confocal microscopy in HeLa cells treated with CR229. Accordingly, these data demonstrate that the anticancer activity of CR229 is associated with: (i) the down-regulation of cyclins and cyclin-dependent kinase; (ii) the induction of p53, p21, and p16; and (iii) the activation of caspase-3.     

Flavopiridol induces cell cycle arrest and p53-independent apoptosis in non-small cell lung cancer cell lines.

Flavopiridol, a synthetic flavone that inhibits tumor growth in vitro and in vivo, is a potent cyclin-dependent kinase (cdk) inhibitor presently in clinical trials. In the present study, the effect of 100-500 nM flavopiridol on a panel of non-small cell lung cancer cell lines was examined. All express a wild-type retinoblastoma susceptibility protein and lack p16INK4A, and only A549 cells are known to express wild-type p53. During 72 h of treatment, flavopiridol was shown to be cytotoxic to all seven cell lines, as measured by trypan blue exclusion, regardless of whether cells were actively cycling. In most cycling cells, cytotoxicity was preceded or accompanied by cell cycle arrest. Cell death resulted in the appearance of cells with a sub-G1 DNA content, suggestive of apoptosis, which was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by demonstration of cleavage of caspase targets including poly(ADP-ribose) polymerase, p21Waf1, and p27Kip1. At doses at or below 500 nM, maximal cytotoxicity required 72 h of exposure. Although flavopiridol resulted in the accumulation of p53 in A549 cells, flavopiridol-mediated apoptosis was p53 independent because it occurred to the same degree in A549 cells in which p53 was targeted for degradation by HPV16E6 expression. The data indicate that flavopiridol has activity against non-small cell lung cancers in vitro and is worthy of continued clinical development in the treatment of this disease.     

Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3

Arsenic trioxide (As(2)O(3)) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As(2)O(3) in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN-28 were treated with various concentrations (0.1 to 100 microM) of As(2)O(3) for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21(waf1/cip1), c-myc, bcl-2 and bax were detected by Western blotting. Effects of As(2)O(3) on caspase-3 protease activity, its protein concentration and cleavage of poly(ADP)-ribose polymerase (PARP) were also studied. As(2)O(3) inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As(2)O(3) induced apoptosis in AGS cells in a concentration- and time-dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co-incubation with p53 anti-sense oligo-nucleotide suppressed As(2)O(3)-induced intracellular p53 over-expression and apoptosis. As(2)O(3) increased the activity of caspase-3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD-fmk and the specific caspase-3 inhibitor DEVD-fmk partially suppressed As(2)O(3)-induced caspase-3 activation and apoptosis. As(2)O(3) inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over-expression and activation of caspase-3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.     

Dulxanthone A induces cell cycle arrest and apoptosis via up-regulation of p53 through mitochondrial pathway in HepG2 cells

Natural products derived from plants provide a rich source for development of new anticancer drugs. Dulxanthone A was found to be an active cytotoxic component in Garcinia cowa by bioactivity-directed isolation. Studies to elucidate the cytotoxic mechanisms of dulxanthone A showed that dulxanthone A consistently induced S phase arrest and apoptosis in the most sensitive cell line HepG2. Furthermore, p53 was dramatically up-regulated, leading to altered expression of downstream proteins upon dulxanthone A treatment. Cell cycle related proteins, such as cyclin A, cyclin B, cyclin E, cdc-2, p21 and p27 were down-regulated. Some apoptosis correlated proteins were also altered following the drug treatment. Bcl-2 family members PUMA was up-regulated while Bcl-2 and Bax were down-regulated. However, the expression ratio of Bax/Bcl-2 was increased. This resulted in the release of cytochrome C from the mitochondria to the cytosol. Concurrently, Apaf-1 was stimulated with p53 by dulxanthone A. In result, cytochrome C, Apaf-1 and procaspase-9 form an apoptosome, which in turn triggered the activation of caspase-9, caspase-3 and downstream caspase substrates. Lamin A/C and PARP were down-regulated or cleaved, respectively. Moreover, cell cycle arrest and apoptosis in HepG2 cells induced by dulxanthone A were markedly inhibited by siRNA knockdown of p53. In summary, dulxanthone A is an active cytotoxic component of G. cowa. It induces cell cycle arrest at lower concentrations and triggers apoptosis at higher concentrations via up-regulation of p53 through the intrinsic mitochondrial pathway in HepG2 cells. Dulxanthone A is therefore likely a promising preventive and/or therapeutic agent against Hepatoma.     

Dihydroartemisinin induces apoptosis in colorectal cancer cells through the mitochondria-dependent pathway

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, has been shown to exhibit antitumor activity in various cancer cells, including colorectal cancer. However, the detailed mechanisms underlying its antitumor activity in colorectal cancer remain to be elucidated. In the present study, we investigated DHA-induced apoptosis in human colorectal cancer HCT-116 cells in vitro. The results showed that DHA treatment significantly reduced cell viability in a concentration- and time-dependent manner. Furthermore, DHA induced G1 cell cycle arrest, apoptotic cell death, and accumulation of reactive oxygen species (ROS). We also found that DHA decreased the mitochondrial membrane potential; activated the caspase-3, caspase-8, and caspase-9; and increased the ratio of Bax/Bcl-2. Meanwhile, the translocation of apoptotic inducing factor (AIF) and the release of cytochrome c from the mitochondria were observed. Strikingly, the free radical scavenger N-acetylcysteine or the caspase-3 inhibitor Ac-DEVD-CHO significantly prevented DHA-induced apoptotic cell death. Taken together, we concluded that DHA-triggered apoptosis in HCT-116 cells occurs through the ROS-mediated mitochondria-dependent pathway. Our data suggest that DHA has great potential to be developed as a novel therapeutic agent for the treatment of human colorectal cancer.     

Diallyl trisulfide induces apoptosis in human primary colorectal cancer cells

Colorectal cancer (CRC) is one of the most prevalent types of cancer worldwide and a common cause of morbidity and mortality in humans. The garlic-derived organosulfur compound diallyl trisulfide (DATS) has been shown to induce apoptosis in many human cancer cell lines in?vitro and also affords significant protection against cancer in animal tumor models in?vivo. There is no available information to show DATS-induced apoptosis in?vitro and the molecular mechanisms of apoptosis in human primary colorectal cancer cells. In this study, we investigated the cytotoxic effects in DATS in primary colorectal cancer cells. DATS inhibited the viability of primary colorectal cancer cells in a time- and dose-dependent manner. After treatment with DATS, primary colorectal cancer cells exhibited DNA condensation by DAPI stain. DATS increased reactive oxygen species (ROS) production in primary colorectal cancer cells. The mitochondria-dependent apoptotic signaling pathway was shown to be involved as determined by increase in the levels of cytochrome c, Apaf-1, AIF and caspase-3 and caspase-9 in DATS-treated primary colorectal cancer cells. The decrease in the level of ΔΨm was associated with an increase in the Bax/Bcl-2 ratio which led to activation of caspase-9 and -3. Based on our results, DATS induces apoptotic cell death in human primary colorectal cancer cells through a mitochondria-dependent signaling pathway.     

Cucurbitacin D induces growth inhibition, cell cycle arrest, and apoptosis in human endometrial and ovarian cancer cells

Cucurbitacin D, a newly isolated triterpenoid cucurbitacin, has been found to possess anticancer effects. The purpose of this study was to elucidate the effects of cucurbitacin D on human endometrial and ovarian cancer cells. Human endometrial and ovarian cancer cells were treated with various concentrations of cucurbitacin D, and its effects on cell growth, the cell cycle, apoptosis, and their related measurements were investigated in vitro. All endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of cucurbitacin D. Cell cycle analysis indicated that their exposure to cucurbitacin D increased the proportion in the sub-G0/G1 phases and G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Our results suggest that cucurbitacin D might be a new therapeutic option for the treatment of endometrial and ovarian cancers.     

顺铂诱导宫颈癌SiHa细胞周期阻滞及凋亡的研究

目的:探讨顺铂在体外对宫颈癌SiHa细胞周期和凋亡的影响及其作用机制。方法:采用MTT法测定顺铂对SiHa细胞增殖的影响;DNAladder和Hoechst33258检测药物作用前后的细胞凋亡情况;流式细胞仪检测药物作用前后的周期阻滞和凋亡;Westernblot检测E6、p53和p21的表达。结果:顺铂抑制SiHa细胞生长呈时效和量效关系,10mol/L顺铂作用SiHa细胞12、24、36和48h,亚G1峰分别为(3·07±0·15)%、(5·18±0·28)%、(17·73±1·20)%和(32·55±2·74)%,与对照组比较差异均有统计学意义,t值分别为32·80、31·46、29·73和25·99,P值均为0·000;顺铂能使SiHa细胞发生G2~M期阻滞,24h达到最高(66·43±4·62)%,凋亡开始;Hoechst33258实验组可见凋亡细胞;琼脂糖凝胶电泳出现DNA梯形条带;Westernblot提示,顺铂作用SiHa细胞后HPVE6的蛋白水平表达逐渐降低,p53和p21蛋白水平表达逐步升高。结论:顺铂随着药物浓度和作用时间的增加对SiHa细胞的体外抑制效应增加,顺铂通过引起SiHa细胞G2~M周期阻滞及抑制HPVE6蛋白的表达,恢复p53功能,启动凋亡,起到杀伤肿瘤作用。     

Rapamycin induces p53-independent apoptosis through the mitochondrial pathway in non-small cell lung cancer cells

The mammalian target of rapamycin (mTOR) is a key kinase acting downstream of growth factor receptor PI3K and AKT signaling, leading to processes resulting in increased cell size and proliferation through translation control. Rapamycin, a specific inhibitor of mTOR, results predominately in G1 cell cycle arrest through translation control and occasionally, cell type-dependent apoptosis by an unknown mechanism. In this study, we investigated the effect and mechanism of action of rapamycin on non-small cell lung cancer (NSCLC) cell lines with p53 mutations. Cell proliferation was evaluated by modified MTT assay. The apoptotic effect of rapamycin was measured by caspase-3 activation and flow cytometric analysis of Annexin V binding. The expression of Bcl-2 and the release of cytochrome?c from mitochondria were evaluated by western blotting. We found that rapamycin induced apoptosis in NSCLC cell lines with p53 mutations. Western blot analysis demonstrated that rapamycin downregulates the expression levels of Bcl-2, which leads to increased cytochrome c release from mitochondria and subsequent activation of caspase cascades. These findings suggest that rapamycin induces p53-independent apoptosis through downregulation of Bcl-2 and the mitochondrial pathway in NSCLC cell lines as a novel antitumor mechanism.